Triage Into the Community for COVID-19 (TICC-19) Patients Pathway - Service evaluation of the virtual monitoring of patients with COVID pneumonia.

INTRODUCTION: COVID-19 pneumonia presented a unique problem for healthcare systems with the potential to overwhelm hospitals and lead to unnecessary morbidity and mortality. Safe triage and follow up systems are required to manage this unprecedented demand. METHODS: We designed a pathway for the triage and assessment of patients based on their resting oxygen saturations and response to a 30 metre rapid walking test. We admitted patients to a 'Virtual Ward' for remote oximetry monitoring from the Emergency Department, step down from inpatient wards and from the local Primary Care 'Hot Hub'. This allowed the safe and managed readmission of those patients who deteriorated at home. RESULTS: During the first wave of COVID-19 we entered 273 onto the pathway for Virtual Ward follow up. Of these, 31 patients were readmitted to hospital, two were admitted to Intensive Care and one patient died. Median oxygen saturation at presentation was 97 % (IQR 96-98%) and following a 30 metre walk test 96% (IQR 94-97%). Median NEWS-2 score was 2 (IQR 1-3). On feedback 99.5% of patients were likely or extremely likely to recommend the service to their family and friends. There was a cost avoidance of £107,600 per month. CONCLUSION: It is safe, feasible and cost effective to set up a triage system with remote oximetry monitoring for patients with COVID-19 and overwhelmingly patients find it a positive experience.

HLA-DR is aberrantly expressed at feto-maternal interface in pre-eclampsia.

In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and Class II molecules, while invasive extravillous trophoblast only express class I HLA-C and the atypical class Ib antigens, HLA-G, -E and -F. Inadequate maternal tolerance of invasive trophoblast has been proposed as a possible immunologic trigger of poor trophoblast invasion and subsequent occurrence of pre-eclampsia. This study aimed to investigate possible aberrant expression of class II HLA-DR on placentae and syncytiotrophoblast-derived extracellular vesicles (STEVs), obtained by dual placental perfusion, from pre-eclampsia (n = 23) and normal pregnant (n = 14) women. Here we demonstrate that HLA-DR can be detected in syncytiotrophoblast from a significant proportion of pre-eclampsia but not control placentae. HLA-DR was also observed, by flow cytometry, on STEVs and associated with placental alkaline phosphatase to validate their placental origin. HLA-DR positive syncytiotrophoblast was detected in placental biopsies from pre-eclampsia but not normal control cases, using immunohistochemistry. The HLA may be fetal or maternal origin. In the latter case a possible mechanism of acquisition is trogocytosis.

Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

Optimising sample collection for placental research.

Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research.

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

Review: Does size matter? Placental debris and the pathophysiology of pre-eclampsia.

A variety of 'debris' is shed from the syncytial surface of the human placenta ranging from large deported multinuclear fragments to sub-cellular components. It is increasingly clear that at least some of this material has signalling functions. Many categories of circulating debris are increased in pre-eclampsia, and exhibit proteins that are pro-inflammatory and could contribute to the systemic inflammatory response in normal pregnancy, which is exaggerated in pre-eclampsia. It is now evident that there is a large 'hidden' population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size. We have used a new technology, nanoparticle tracking analysis, to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion. The vesicles range in size from 50 nm to 1 µm with the majority being <500 nm (which includes both exosomes and microvesicles). We speculate whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in the maternal syndrome of pre-eclampsia.

OS045. Multi-dimensional protein identification technology analysis of syncytiotrophoblast vesicles released from perfused preeclampsia placentas.

INTRODUCTION: In pre-eclampsia, the consequences of poor placentation lead to the second stage of pre-eclampsia, which involves activation of a maternal systemic inflammatory response (MSIR). Endothelial and other inflammatory cellular dysfunction cause the diverse features which characterise the disorder. We have previously shown that syncytiotrophoblast microvesicles (STBM) are pro-inflammatory and circulate in increased amounts in pre-eclamptic women. We hypothesise that multiple placental "danger signals" are carried by STBM into the maternal circulation in increased amounts in PE with pro-inflammatory, anti-angiogenic and pro-coagulant activity, implicating STBM in the pathophysiology of PE. OBJECTIVES: To characterise the proteins carried by STBM from normal and PE placentas. For the first time multi-dimensional protein identification technology (MudPIT) was used to derive the proteome profiles of normal and PE placenta STBM. METHODS: STBM were prepared from placentas (normal term: n=9 and PE: n=5) by dual lobe perfusion, isolated by ultracentrifugation and stored at -80°C. Normal and PE derived placenta STBM pools were then subjected to MudPIT analysis. RESULTS: 538 proteins unique to PE STBM, 604 proteins unique to normal STBM and 1421 proteins common to both preparations were found. Preliminary analysis indicates the presence of alarmins (HSP70, and galectin 3), exosomal proteins (CD63,CD9,CD81), immunoregulatory molecules (CD26,CD200,CD47,Galectin 1), complement and complement regulatory molecules (C1q,C3,CD55,CD59 and vitronectin), amino acid transporters (CD98) and anti-angiogenic molecules (endoglin). Our analysis also reveals that proteins known to be elevated in blood before, or at, the time of pre-eclampsia are elevated or unique in STBM from PE placentas, including Fetuin A, Inter-alpha (globulin) inhibitor H4, Serum amyloid P component, Apolipoprotein H (or B2GP1) and Apolipoprotein AII. Thus, as predicted, a large number of circulating molecules are associated with STBM. The inter-relationships between proteins that are unique to either PE or normal pregnancy and the processes in which they are involved are being determined by Ingenuity Pathways Analysis software. In terms of biofunctions, preliminary analysis shows that proteins unique to PE STBM have a highly significant association (p<10(-11)) with 6 disease pathways including inflammatory, immunological, cardiovascular and reproductive system diseases and organ injury, whereas for proteins unique to normal STBM only protein synthesis was significant at the same level. CONCLUSION: STBM contain a heterogeneous population of vesicles that convey a large repertoire of placental proteins into the maternal circulation. The profound differences between PE and normal STBM indicate their pro-inflammatory potential.

Vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast.

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Caveolin-1 and lipid rafts in confluent BeWo trophoblasts: evidence for Rock-1 association with caveolin-1.

Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Galphaq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell-cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.

Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells.

OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from the cultures and analysed on microarrays. SETTING: A laboratory investigation using placentas obtained from a hospital delivery ward. SAMPLE: Placentas from nine healthy women were obtained. STBM vesicles were isolated from the placentas and umbilical vein endothelial cell cultures were established from the umbilical cords. METHODS: Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Fold changes in gene expression levels between treated and control cultures were calculated from the microarray results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation of endothelial cells by exposure to STBM. The unfolded protein response in particular may be involved. CONCLUSIONS: STBM may influence endothelial cell function during pregnancy but STBM alone cannot account for the entire range of endothelial dysfunctions observed in pre-eclampsia.

Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction.

RATIONALE: Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive intrauterine growth restriction (nIUGR), but in the absence of maternal disease. OBJECTIVE: To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia. METHODS: A prospective case-control study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET < 34 week), 2) women with late-onset pre-eclampsia (LOPET > or = 34 week), 3) women with nIUGR), 4) matched normal pregnant women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p < 0.05. RESULTS: STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median): 41 ng/ml, n = 15) compared with matched normal pregnancy (16 ng/ml, n = 15; Wilcoxon p = 0.005). LOPET (50 ng/ml, n = 10) and nIUGR (18 ng/ml, n = 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, n = 15, and 36 ng/ml, n = 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, n = 10. CONCLUSIONS: Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the pathogenesis of the maternal syndrome.

Endothelial cells and peripheral blood mononuclear cells are a potential source of extraplacental activin a in preeclampsia.

An excessive systemic inflammatory response, involving endothelial cells and leukocytes, underlies the maternal symptoms of preeclampsia. Activin A is raised in preeclampsia, suggesting a possible involvement in its pathophysiology. The placenta is the main source of activin A in normal pregnancy. We investigated whether peripheral blood mononuclear cells (PBMCs) and endothelium, activated by proinflammatory stimuli, were a potential source of activin A in preeclampsia. Both endotoxin and TNFalpha stimulated activin A secretion by PBMCs from nonpregnant, preeclamptic, and matched normal pregnant women (P < 0.05). Pregnancy increased the responsiveness of PBMCs to endotoxin (P < 0.05), whereas only the preeclamptic group were significantly more responsive to TNFalpha (P < 0.05). Human umbilical vein endothelial cells secreted activin A spontaneously and in response to TNFalpha (P < 0.05), but recombinant IL-1beta and IL-6 had no significant effect over the 72-h culture period. Inhibin A and follistatin were undetectable (<2 pg/ml and < 20 pg/ml, respectively) in PBMCs and human umbilical vein endothelial cell culture media. These data suggest that PBMCs and endothelium, activated by TNFalpha, could be extraplacental sources of activin A in preeclampsia. The pathological significance of increased activin A in preeclampsia is unknown, although it may have a role in the mechanisms underlying endothelium dysfunction.

Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells.

The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P(4) output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number ( approximately 1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output ( approximately 2.5-fold) and cell number ( approximately 2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio ( approximately 3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.

Measurement of inhibin-A (alpha beta A dimer) during the oestrous cycle, after manipulation of ovarian activity and during pregnancy in ewes.

A new two-site ELISA was validated for ovine plasma and used to measure circulating inhibin-A concentrations during a synchronized oestrous cycle in four ewes and throughout pregnancy in six ewes. Inhibin A concentrations were also determined in four ewes during chronic treatment with a GnRH agonist and after subsequent exposure to pregnant mares' serum gonadotrophin (PMSG) to stimulate ovarian follicular development. Concentrations of FSH, LH, oestradiol and progesterone were determined by radioimmunoassay. The detection limit of the inhibin-A ELISA was approximately 50 pg ml-1 and no significant crossreaction was observed with a range of related molecules including activin-A, inhibin-B, activin-B, follistatin and alpha 2-macroglobulin. Inhibin-A concentrations were below the detection limit in plasma from hypophysectomized and ovariectomized ewes. During the oestrous cycle, plasma inhibin-A concentrations (approximately 0.3-0.4 ng ml-1) did not vary during the follicular phase whereas plasma oestradiol increased approximately tenfold. After the preovulatory LH/FSH surge, inhibin-A fell to a nadir (approximately 0.15 ng ml-1) coincident with the peak of the postovulatory FSH rise. During the next 2 days, FSH concentrations fell to basal values as inhibin-A concentrations increased (P < 0.05) to a peak (approximately 0.5 ng ml-1) 3 days after the preovulatory LH/FSH surge. Over the following 3 days, FSH values increased again (P < 0.05) as inhibin-A concentrations fell to approximately 0.25 ng ml-1 (P < 0.05). Chronic GnRH agonist treatment suppressed FSH concentrations by about 50%, while inhibin-A and oestradiol concentrations fell below detection limits. Within 2 days after the PMSG injection, concentrations of inhibin-A (approximately 4.5 ng ml-1) and oestradiol (approximately 20 pg ml-1) had increased to very high values, while FSH concentrations had been reduced by a further 50%. Plasma concentrations of inhibin-A and FSH were similar to those in nonpregnant ewes during the first 60 days of gestation, but inhibin-A values fell markedly (sevenfold; P < 0.01) between days 60 and 90, coincident with a twofold decrease in FSH (P < 0.05). Inhibin A and FSH concentrations remained low for the remainder of gestation and were positively correlated throughout pregnancy (r = 0.48; P < 0.005). These observations support an endocrine feedback role for ovarian inhibin-A and oestradiol in controlling the secondary (postovulatory) FSH surge in ewes, but indicate that an increase in oestradiol is responsible for the characteristic reduction in FSH during the early to mid-follicular phase. The reduced secretion of FSH from mid- to late pregnancy cannot be attributed to increased inhibin-A secretion by the feto-placental unit, but most likely reflects increased steroid secretion from this source.

Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n = 6) and control (CON; n = 8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles > or = 3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had approximately 3-fold more follicles > or = 3 mm (P < 0.0001) and approximately 3-fold more oestrogenic follicle (P < 0.001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (approximately 6-fold; P < 0.001) and inhibin A (approximately 3-fold; P < 0.001) but only slightly more follistatin (approximately 1.4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (approximately 1:1) was significantly higher (P < 0.001) than in follicles from CON ewes (approximately 0.3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.

Effects of supplementary treatment with bovine growth hormone on hormonal and ovulatory responses to inhibin immunization in ewes.

The aim of this study was to determine whether supplementary treatment with recombinant bovine growth hormone(rbGH) can enhance the ovulatory response of ewes to inhibin immunization. Crossbred ewes (n = 20) were actively immunized against bovine inhibin a1-29 peptide conjugate while 20 ewes served as controls. Oestrus was synchronized using progestagen sponges and ewes were allocated to four groups: control ewes (n = 10); control ewes given rbGH (n = 10); inhibin-immunized ewes (n = 10) and inhibin-immunized ewes given rbGH (n = 10). A single s.c. dose of rbGH (50 mg) was given 7 days before sponge removal. Blood was collected for measurement of inhibin antibody titre, and concentrations of insulin-like growth factor I (IGF-I), FSH, oestradiol and progesterone. Ovulation, pregnancy and lambing rates were also recorded. All inhibin-immunized ewes produced antibodies that bound 125I-labelled (32 kDa) inhibin. The concentration of FSH in the plasma of the ewes after the second booster inhibin immunization was higher than that in control ewes (P < 0.005). Treatment with rbGH promoted a 2-3-fold increase in plasma concentration of IGF-I (P < 0.001); the response was less (P < 0.01) in immunized compared with control ewes. Treatment with rbGH alone had no significant effect on the concentration of FSH or oestradiol or on ovulation rate or litter size. Overall, inhibin-immunized ewes had higher mean FSH concentrations (P < 0.002), higher preovulatory oestradiol surges (P < 0.05) and higher progesterone concentrations in the luteal phase (P < 0.0001). Treatment with rbGH reduced the effects of immunization on FSH (P < 0.01) and progesterone (P < 0.02) concentrations. Immunized ewes showed a threefold increase in ovulation rate (P < 0.001) and a 1.8-fold increase in litter size (P < 0.05) compared with control ewes. In immunized ewes given rbGH, ovulation rate was increased by a factor of 2.2 and litter size by a factor of 1.8. In conclusion, these data do not support the hypothesis that supplementary treatment of ewes with rbGH to raise plasma IGF-I concentrations (and presumably intraovarian IGF-I) can enhance the ovulatory response to inhibin immunization.