Diagnostic utility of clinicodemographic, biochemical and metabolite variables to identify viable pregnancies in a symptomatic cohort during early gestation.

A significant number of pregnancies are lost in the first trimester and 1-2% are ectopic pregnancies (EPs). Early pregnancy loss in general can cause significant morbidity with bleeding or infection, while EPs are the leading cause of maternal mortality in the first trimester. Symptoms of pregnancy loss and EP are very similar (including pain and bleeding); however, these symptoms are also common in live normally sited pregnancies (LNSP). To date, no biomarkers have been identified to differentiate LNSP from pregnancies that will not progress beyond early gestation (non-viable or EPs), defined together as combined adverse outcomes (CAO). In this study, we present a novel machine learning pipeline to create prediction models that identify a composite biomarker to differentiate LNSP from CAO in symptomatic women. This prospective cohort study included 370 participants. A single blood sample was prospectively collected from participants on first emergency presentation prior to final clinical diagnosis of pregnancy outcome: LNSP, miscarriage, pregnancy of unknown location (PUL) or tubal EP (tEP). Miscarriage, PUL and tEP were grouped together into a CAO group. Human chorionic gonadotrophin ß (ß-hCG) and progesterone concentrations were measured in plasma. Serum samples were subjected to untargeted metabolomic profiling. The cohort was randomly split into train and validation data sets, with the train data set subjected to variable selection. Nine metabolite signals were identified as key discriminators of LNSP versus CAO. Random forest models were constructed using stable metabolite signals alone, or in combination with plasma hormone concentrations and demographic data. When comparing LNSP with CAO, a model with stable metabolite signals only demonstrated a modest predictive accuracy (0.68), which was comparable to a model of ß-hCG and progesterone (0.71). The best model for LNSP prediction comprised stable metabolite signals and hormone concentrations (accuracy = 0.79). In conclusion, serum metabolite levels and biochemical markers from a single blood sample possess modest predictive utility in differentiating LNSP from CAO pregnancies upon first presentation, which is improved by variable selection and combination using machine learning. A diagnostic test to confirm LNSP and thus exclude pregnancies affecting maternal morbidity and potentially life-threatening outcomes would be invaluable in emergency situations.

The dynamic changes in the number of uterine natural killer cells are specific to the eutopic but not to the ectopic endometrium in women and in a baboon model of endometriosis.

BACKGROUND: Endometriosis is a common condition associated with growth of endometrial-like tissue beyond the uterine cavity. Previous reports have suggested a role for uNK cells in the pathogenesis of endometriosis postulating that survival and accumulation of menstrual endometrial tissue in the peritoneal cavity may relate to a reduction in the cytotoxic activity of peripheral blood NK cells. We aimed to assess the differences in percentage of uNK cells and their phenotypical characterization in eutopic and ectopic endometrial samples from women with and without endometriosis and baboons with induced endometriosis. METHODS: Eutopic and ectopic endometrial samples from 82 women across the menstrual cycle with/without endometriosis and from 8 baboons before and after induction of endometriosis were examined for CD56 and NKp30 expression with immunohistochemistry, quantified using computer assisted image analysis. Curated secretory phase endometrial microarray datasets were interrogated for NK cell receptors and their ligands. In silico data was validated by examining the secretory phase eutopic endometrium of women with and without endometriosis (n = 8/group) for the immuno-expression of BAG6 protein. RESULTS: The percentage of uNK cells increased progressively from the proliferative phase with the highest levels in the late secretory phase in the eutopic endometrium of women with and without endometriosis. The percentage of uNK cells in ectopic lesions remained significantly low throughout the cycle. In baboons, induction of endometriosis increased the percentage of uNK in the ectopic lesions but not NKp30. Published eutopic endometrial microarray datasets demonstrated significant upregulation of NKp30 and its ligand BAG6 in women with endometriosis compared with controls. Immunohistochemical staining scores for BAG6 was also significantly higher in secretory phase eutopic endometrium from women with endometriosis compared with the endometrium of healthy women (n = 8/group). CONCLUSIONS: The dynamic increase in the percentage of uNK cells in the secretory phase is preserved in the endometrium of women with endometriosis. The low number of uNK cells in human and baboon ectopic lesions may be due to their exaggerated reduction in hormonal responsiveness (progesterone resistance).

A rapid, reliable method for uNK cell density estimation.

Interest in uterine NK cell density as a diagnostic or screening tool in reproductive disorders is growing. However, current methods of analysis are time consuming. In this study, 997 images from 100 women were analysed both by the currently used method combining manual and computer aided counting, and by using colour splitting combined with area measurement as an estimate of positively stained cells. Good correlation was found between both methods (r(2)=0.92) centred around the line of equality, with no systematic differences observed in Bland Altman plots. The area measurement was significantly faster and thus is a useful screening method.

A novel method of collection of saliva for estimation of steroid levels in extremely premature infants.

AIM: The major advantage of salivary cortisol sampling is that it is considerably less invasive than taking a blood sample. However, previous methods of obtaining saliva in premature infants have been poorly tolerated and inaccurate. We describe a simple, non-distressing technique for obtaining saliva samples to assess extremely premature infants' salivary cortisol status. METHODS: We prospectively obtained early morning saliva samples from extremely premature infants. Their gestational age ranged between 23 and 27 weeks. Saliva was obtained using four standard universal swabs by placing one swab at a time in the infant's mouth for 1-2 min. No salivary stimulants were used. RESULTS: There were 65 infants (36 males). Mean gestation was 25.3 ± 1.3 weeks. This technique had a success rate of 85% in obtaining a mean of 150 µL of saliva (range 50-350 µL) by trained staff. No adverse events were recorded. CONCLUSION: We describe a novel, safe, non-distressing and effective method of saliva collection for salivary cortisol measurement in extremely premature infants.

Localization of angiogenic growth factors and their receptors in the human endometrium throughout the menstrual cycle and in recurrent miscarriage.

BACKGROUND: Angiogenesis is a key feature of endometrial development. Inappropriate endometrial vascular development has been associated with recurrent miscarriage (RM) with increased amounts of perivascular smooth muscle cells surrounding them. METHODS: In the current study, we have used immunohistochemistry to study temporal and spatial expression of a series of angiogenic growth factors (AGFs) and their receptors; vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, platelet-derived growth factor (PDGF)-BB, PDGF-Rα, PDGF-Rß, transforming growth factor (TGF)-ß1, TGF-ßRI, TGF-ßRII, angiopoietin (Ang)-1, Ang-2 and Tie-2, in the proliferative, early secretory and mid-late secretory phase endometrium from control women as well as in the mid-late secretory phase of women with a history of RM. The AGFs and their receptors studied were immunostained and assessed separately in stromal, vascular smooth muscle, endothelial and glandular epithelial cells. Laser capture microdissection and real-time RT-PCR were used to confirm expression patterns observed by immunohistochemistry. RESULTS: Most AGFs investigated showed both temporal and spatial expression patterns in normal cycling endometrium. In addition, immunostaining intensity for several AGFs was altered in women with a history of RM, particularly in vascular smooth muscle cells (VSMCs). VSMC expression of TGF-ß1, VEGF-R1 and VEGF-R2 was increased while expression of PDGF-BB, TGF-ßRI, TGF-ßRII, Ang-2, VEGF-A and VEGF-C was reduced. CONCLUSIONS: This study confirms that the cycling endometrium is a highly angiogenic tissue and that this process is likely to be altered in women with a history of RM and may contribute to the aetiology of this condition.

Endometrial cell counts in recurrent miscarriage: a comparison of counting methods.

AIMS: Studies of uterine natural killer (uNK) cells require reliable measurements of uNK cell density among diverse endometrial tissue. The aim of this study was to compare cell counting manually with two computer-aided methods based on a public domain software package, ImageJ. METHODS AND RESULTS: Immunohistochemistry (IHC) of CD56(+) uNK cells was performed on endometrium from recurrent miscarriage patients. Numbers of stromal cells per high-power field (HPF) were counted by two observers using: (i) manual tally counter and graticule; (ii) ImageJ 'point picker' tool; and (iii) ImageJ 'particle analysis' tool. Coefficients of variation (CV) and Bland-Altman plots were used to evaluate interobserver differences. Evaluation of %uNK using ImageJ particle analysis for stromal cell counts and point picker tool for uNK counts was undertaken. Point picker and particle analysis were significantly better than manual counting [interobserver CVs mean (standard deviation) 6.1% (3.3%); 4.7% (3.9%), 8.2% (6.5%), respectively]. Mean inter- and intra-observer CVs for %uNK were 10.3% (6.6%), 8.5% (4.9%) and 6.8% (4.3%), respectively. Bland-Altman analysis revealed no systematic differences in cell counts with the number of cells in the image for each method. CONCLUSIONS: Compared to manual cell counting, computer-aided image analysis yields more reproducible results for the assessment of uNK cells density using IHC.

Prednisolone treatment reduces endometrial spiral artery development in women with recurrent miscarriage.

BACKGROUND: Uterine natural killer (uNK) cells and endometrial blood vessel maturation are increased in the luteal phase of the menstrual cycle in a subset of women with recurrent miscarriage (RM). uNK cell numbers are reduced after treatment with prednisolone (20 mg/day for 3 weeks). HYPOTHESES: Prednisolone treatment reduces endometrial vascular maturation and angiogenic growth factor expression in women with RM with increased uNK cells. METHODS: Endometrial biopsies (n = 18 paired samples) from women with RM at LH + 7 before and during prednisolone treatment (20 mg/day for 3 weeks) were snap frozen. Total RNA and cDNA was prepared and used in a human angiogenesis RT-PCR superarray (84 genes, n = 6 pairs) with results validated using RT-PCR (n = 15 pairs). Immunohistochemistry (n = 15 pairs) was performed for Factor VIII, α-smooth muscle actin (α-SMA) and myosin heavy chain (MyHC) and the total number of vessels and the percentage of vessels completely surrounded by vascular smooth muscle cells (VSMCs) were determined. RESULTS: During prednisolone treatment there was no change in the total number of endometrial blood vessels but the percentage of vessels completely surrounded by VSMCs was decreased (α-SMA P < 0.0001; MyHC P < 0.0001). Endometrial EGF and STAB 1 expression was decreased during prednisolone treatment in samples from woman who went on to have a live birth. CONCLUSIONS: The effect of prednisolone therapy for some women with RM may be due to altered endometrial angiogenic growth factor expression and reduced blood vessel maturation.

Reduction of oxidative stress marker in lung fluid of preterm infants after administration of intra-tracheal liposomal glutathione.

BACKGROUND: Low levels of glutathione are associated with subsequent chronic lung disease in preterm infants. Incorporation of glutathione into liposomes offers a method of increasing levels with a prolonged half-life compared with direct inhalation. OBJECTIVES: The aim of this study was to examine the clinical feasibility of administering a single dose of liposomal glutathione and its effectiveness at raising glutathione at 12 and 24 h after treatment. METHODS: Fourteen ventilated preterm infants from the Regional Neonatal Intensive Care Unit at Liverpool Women's Hospital received 1 mg/kg or 10 mg/kg liposomal glutathione intra-tracheally and bronchoalveolar lavage fluid was collected prior to treatment, 12 and 24 h after dosing for glutathione and malondialdehyde estimation. RESULTS: Mean glutathione was initially 12.2 micromol/l, increasing to 52.8 micromol/l at 12 h (p = 0.006). Mean malondialdehyde was initially 265.6 nmol/l decreasing to 11.2 nmol/l at 12 h (p = 0.018). CONCLUSIONS: Intra-tracheal liposomal glutathione instillation offers a feasible method of raising pulmonary glutathione in preterm infants and shows biochemical antioxidant effects.