A systematic review identifying seminal plasma biomarkers and their predictive ability on IVF and ICSI outcomes.

The diverse nature and high molecule concentration of seminal plasma (SP) makes this fluid a good potential source for a potential biomarker that could predict assisted reproductive technology (ART) outcomes. Currently, semen quality parameters cannot accurately predict ART outcomes. A systematic literature search was conducted to identify human SP biomarkers with potential predictive ability for the outcomes of IVF and intracytoplasmic sperm injection. Observational cohort and case-control studies describing the association between biomarkers in human SP and the outcome of infertile men attending for ART were included. Forty-three studies were selected, reporting on 89 potential SP biomarkers (grouped as oxidative stress, proteins glycoproteins, metabolites, immune system components, metals and trace elements and nucleic acids). The present review supports 32 molecules in SP as potentially relevant biomarkers for predicting ART outcomes; 23 molecules were reported once and nine molecules were reported in more than one study; IL-18 and TGF-ß1-IL-18 ratio were confirmed in distinct studies. This review presents the most comprehensive overview of relevant SP biomarkers to predict ART outcomes to date, which is of clinical interest for infertility investigations and assisted reproduction; nevertheless, its potential is under-exploited. This review could serve as starting point for designing an all-encompassing study for biomarkers in SP and their predictive ability for ART outcomes, and for developing a non-invasive diagnostic tool.

Oncofertility care for newly diagnosed girls with cancer in a national pediatric oncology setting, the first full year experience from the Princess Máxima Center, the PEARL study.

BACKGROUND: Childhood cancer patients often remain uninformed regarding their potential risk of gonadal damage. In our hospital we introduced a five step standard oncofertility care plan for all newly diagnosed female patients aiming to identify, inform and triage 100% of patients and counsel 100% of patients at high risk (HR) of gonadal damage. This observational retrospective study (PEARL study) evaluated the use of this standard oncofertility care plan in the first full year in a national cohort. METHODS: The steps consist of 1)timely (preferably before start of gonadotoxic treatment) identification of all new patients, 2)triage of gonadal damage risk using a standardized gonadal damage risk stratification tool, 3)informing all patients and families, 4)counseling of a selected subset of girls, and 5) fertility preservation including ovarian tissue cryopreservation (OTC) in HR patients using amended Edinburgh criteria. A survey of the medical records of all girls newly diagnosed with cancer the first year (1-1-2019 until 31-12-2019) was conducted. RESULTS: Of 261 girls, 228 (87.4%) were timely identified and triaged. Triage resulted in 151 (66%) low(LR), 32 (14%) intermediate(IR) and 45 (20%) high risk(HR) patients. Ninety-nine families were documented to be timely informed regarding gonadal damage risk. In total, 35 girls (5 LR, 5 IR, 25 HR) were counseled by an oncofertility expert. 16/25 HR patients underwent fertility preservation (1 ovariopexy + OTC, oocyte cryopreservation (1 with and 1 without OTC) and 13 OTC). Fertility preservation did not lead to complications or delay of cancer treatment in any patient. CONCLUSION: We timely identified and triaged most girls (88%) with cancer with a high risk of gonadal damage to be counseled for fertility preservation. We aim to optimize the oncofertility care plan and the standardized gonadal damage risk stratification tool based on this experience and these may be of value to other pediatric oncology centers.

Comparing the cumulative live birth rate of cleavage-stage versus blastocyst-stage embryo transfers between IVF cycles: a study protocol for a multicentre randomised controlled superiority trial (the ToF trial).

INTRODUCTION: In vitro fertilisation (IVF) has evolved as an intervention of choice to help couples with infertility to conceive. In the last decade, a strategy change in the day of embryo transfer has been developed. Many IVF centres choose nowadays to transfer at later stages of embryo development, for example, transferring embryos at blastocyst stage instead of cleavage stage. However, it still is not known which embryo transfer policy in IVF is more efficient in terms of cumulative live birth rate (cLBR), following a fresh and the subsequent frozen-thawed transfers after one oocyte retrieval. Furthermore, studies reporting on obstetric and neonatal outcomes from both transfer policies are limited. METHODS AND ANALYSIS: We have set up a multicentre randomised superiority trial in the Netherlands, named the Three or Fivetrial. We plan to include 1200 women with an indication for IVF with at least four embryos available on day 2 after the oocyte retrieval. Women are randomly allocated to either (1) control group: embryo transfer on day 3 and cryopreservation of supernumerary good-quality embryos on day 3 or 4, or (2) intervention group: embryo transfer on day 5 and cryopreservation of supernumerary good-quality embryos on day 5 or 6. The primary outcome is the cLBR per oocyte retrieval. Secondary outcomes include LBR following fresh transfer, multiple pregnancy rate and time until pregnancy leading a live birth. We will also assess the obstetric and neonatal outcomes, costs and patients' treatment burden. ETHICS AND DISSEMINATION: The study protocol has been approved by the Central Committee on Research involving Human Subjects in the Netherlands in June 2018 (CCMO NL 64060.000.18). The results of this trial will be submitted for publication in international peer-reviewed and in open access journals. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL 6857).

Recombinant hepatocyte growth factor treatment in a canine model of congenital liver hypoplasia.

BACKGROUND: Although the liver has a large regenerative capacity, in many hepatopathies, these repair mechanisms fail. The therapeutic potential of hepatocyte growth factor (HGF) has been proven in numerous toxin-induced liver failure models in rodents, but never in spontaneously occurring liver diseases in larger animal models. AIM: The aim of this study was to induce liver growth in a hypoplastic liver by the administration of exogenous recombinant HGF. The natural hypoplastic liver model used is the canine congenital portosystemic shunt (CPSS) characterized by strongly reduced liver growth and function. METHODS: Recombinant HGF (rHGF), 200 µg/kg, was given twice daily during 3 weeks by an intravenous injection in six dogs with CPSS. Liver volumes were determined by computed tomography before and at 1, 2, 3 and 7 weeks after the initiation of treatment. Portosystemic shunting was evaluated with an ammonia tolerance test and liver portal perfusion was quantified with scintigraphy. Simultaneously, blood parameters for liver function were assayed and liver biopsies were taken for histology, immunohistochemistry and gene-expression measurements. RESULTS: During 3 weeks of HGF treatment, hepatocyte proliferation increased and an increase in liver volume up to 44% was seen, persisting in two dogs up to 4 weeks after the termination of treatment. Ki-67 expression, gene expression of E2F1 and CDC6, phosphorylated-c-MET and phosphorylated-ERK1/2 protein levels confirmed increased hepatocyte proliferation and HGF signalling. The aberrant portal perfusion did not change during treatment. CONCLUSIONS: Transient in vivo liver growth is shown using CPPS as a naturally occurring large animal model, indicating the therapeutic potential of HGF in liver disease.

The dog liver contains a "side population" of cells with hepatic progenitor-like characteristics.

The aim of this study was to isolate and characterize potential progenitor cells from healthy dog livers. Stem/progenitor cells can be prospectively isolated from a diversity of tissues using their ability to efficiently pump out the dye Hoechst33342, thereby portraying a side population (SP) in dual-wavelength flow cytometry. We here describe the detection of a SP in dog liver, constituting approximately 3 % of the nonparenchymal-enriched cell fractions. A subpopulation of the SP (approximately 30 %) was immunonegative for the panhematopoietic marker CD45, and consisted predominantly of small, mononuclear, keratin 7-immunoreactive cells; characteristics suggestive of a liver progenitor cell phenotype. Both the CD45- and CD45+ SP showed upregulated expression of progenitor/cholangiocyte marker genes, but also low-level expression of hepatocyte markers, suggesting the presence of progenitor cells committed to the hepatic lineage in both SP fractions. Our findings demonstrate that healthy canine liver contains a small population of cells with progenitor-like characteristics that can be isolated on the basis of efficient Hoechst33342 expulsion.

In vitro differentiation of liver progenitor cells derived from healthy dog livers.

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor.

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.

Hepatocyte growth factor improves viability after H2O2-induced toxicity in bile duct epithelial cells.

Intracellular defence mechanisms against oxidative stress may play an important role in the progression of liver diseases, including cholangiopathies. The multifunctional anti-apoptotic hepatocyte growth factor (HGF) has been suggested to have antioxidant functions. The effect of HGF upon cell viability, the generation of ROS, the expression of genes that play a role in ROS defence, and the activation of caspase-3 were measured in bile duct epithelial (BDE) cells in the presence or absence of H(2)O(2). HGF reduced H(2)O(2)-induced loss of viability, diminished H(2)O(2)-mediated ROS generation and abrogated H(2)O(2)-triggered changes in GSH/GSSG ratio. Furthermore, HGF increased the gene-expression of gamma-glutamylcysteine synthetase (GCLC) and glutathione reductase (GSR), while no effect was seen upon the gene-expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX1), and glutathione synthetase (GSR). Finally, HGF diminished the proteolytical activation of the key mediator of apoptosis (caspase-3) after H(2)O(2) exposure. Together, HGF may improve viability in bile duct epithelia cells after H(2)O(2) induced toxicity by proliferation, strengthening the intrinsic antioxidant defences, and/or by an attenuation of apoptosis. These in vitro results support the evaluation of HGF as antioxidative factor in hepatobiliary pathologies.

Major HGF-mediated regenerative pathways are similarly affected in human and canine cirrhosis.

BACKGROUND: The availability of non-rodent animal models for human cirrhosis is limited. We investigated whether privately-owned dogs (Canis familiaris) are potential model animals for liver disease focusing on regenerative pathways. Several forms of canine hepatitis were examined: Acute Hepatitis (AH), Chronic Hepatitis (CH), Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis), and Cirrhosis (CIRR). Canine cirrhotic samples were compared to human liver samples from cirrhotic stages of alcoholic liver disease (hALC) and chronic hepatitis C infection (hHC). RESULTS: Canine specific mRNA expression of the regenerative hepatocyte growth factor (HGF) signaling pathway and relevant down-stream pathways were measured by semi-quantitative PCR and Western blot (STAT3, PKB, ERK1/2, and p38-MAPK). In all canine groups, levels of c-MET mRNA (proto-oncogenic receptor for HGF) were significantly decreased (p < 0.05). Surprisingly, ERK1/2 and p38-MAPK were increased in CH and LDH. In the human liver samples Western blotting indicated a high homology of down-stream pathways between different etiologies (hALC and hHC). Similarly activated pathways were found in CIRR, hALC, and hHC. CONCLUSION: In canine hepatitis and cirrhosis the major regenerative downstream pathways were activated. Signaling pathways are similarly activated in human cirrhotic liver samples, irrespective of the differences in etiology in the human samples (alcohol abuse and HCV-infection). Therefore, canine hepatitis and cirrhosis could be an important clinical model to evaluate novel interventions prior to human clinical trials.

Copper metabolism and oxidative stress in chronic inflammatory and cholestatic liver diseases in dogs.

Inherited defects of copper metabolism resulting in hepatic copper accumulation and oxidative-stress might cause breed-associated forms of hepatitis. Biliary excretion is the major elimination route of copper, therefore increased hepatic copper concentrations could also be caused by cholestasis. The aim of this study was to find criteria to determine whether copper-accumulation is primary or occurs secondary to hepatitis. Liver samples of Bedlington Terriers with copper toxicosis (CT), breeds with non-copper-associated chronic extrahepatic cholestasis (EC) or chronic hepatitis (CH), and healthy dogs were used. Copper metabolism was analyzed by means of histochemical staining (copper concentration) and quantitative reverse transcriptase polymerase chain reaction (Q-PCR) on copper excretion/storage (ATOX1, COX17, ATP7A, ATP7B, CP, MT1A, MURR1, XIAP). Oxidative stress was measured by determining GSH/GSSG ratios and gene-expression (SOD1, CAT, GSHS, GPX1, CCS, p27KIP, Bcl-2). Results revealed 5+ copper in CT, but no or 1-2+ copper in EC and CH. Most gene products for copper metabolism remained at concentrations similar to healthy dogs. Three clear exceptions were observed in CT: 3-fold mRNA increase of ATP7A and XIAP and complete absence of MURRI. The only quantitative differences between the diseased and the control groups were in oxidative stress, evidenced by reductions in all GSH/GSSG ratios. We conclude that 3+ or higher histochemical detection of copper indicates a primary copper storage disease. The expression profile of copper-associated genes can be used as a reference for future studies on copper-associated diseases. All 3 diseases have reduced protection against oxidative stress, opening a rationale to use antioxidants as possible therapy.

Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin.

BACKGROUND: Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA) directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE), mammary carcinoma (P114), and osteosarcoma (D17). These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR) and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay. RESULTS: All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively. CONCLUSION: XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors.

Transforming growth factor beta-1 signalling in canine hepatic diseases: new models for human fibrotic liver pathologies.

BACKGROUND/AIMS: The purpose of this study was to validate spontaneous chronic hepatitis and cirrhosis in dogs as a potential large animal model for fibrotic liver disease in humans by evaluating their molecular pathophysiology. METHODS: Transforming growth factor-beta1 (TGF-beta1) signalling was analysed in liver samples of dogs with acute hepatitis (AH), chronic hepatitis (CH), cirrhosis (CIRR), and a specific form of cirrhosis, lobular dissecting hepatitis (LDH), in comparison with human cirrhotic samples from alcohol abuse (ALC) and hepatitis C (HC). RESULTS: Canine samples were investigated with quantitative real-time PCR (Q-PCR) and Western blotting on TGF-beta1 signalling including Smad2/3 phosphorylation. Immunohistochemistry on collagens I and III was performed. Q-PCR showed an increase in TGF-beta1 levels and downstream effector gene products in CH, LDH, and CIRR. The same fibrotic diseases also showed an increase in phosphorylated Smad2/3 and a higher deposition of collagens I and III. In contrast, in AH neither active TGF-beta1 signalling nor collagen deposition was observed. Western blot analysis on human ALC and HC indicated a high similarity with canine samples in TGF-beta1 expression and Smad2/3 phosphorylation. CONCLUSIONS: Our results demonstrate that fibrosis in spontaneous dog liver diseases is highly comparable to their human counterparts and might serve as models for anti-fibrotic strategies.

Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment.

BACKGROUND: We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. RESULTS: We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor beta1 (TGF-beta1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-beta1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-beta1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-beta1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. CONCLUSION: The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis.

Differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in Doberman pinschers.

BACKGROUND: The role of copper accumulation in the onset of hepatitis is still unclear. Therefore, we investigated a spontaneous disease model of primary copper-toxicosis in Doberman pinschers so to gain insights into the pathophysiology of copper toxicosis, namely on genes involved in copper metabolism and reactive oxygen species (ROS) defences. RESULTS: We used quantitative real-time PCR to determine differentially expressed genes within a target panel, investigating different groups ranging from copper-associated subclinical hepatitis (CASH) to a clinical chronic hepatitis with high hepatic copper concentrations (Doberman hepatitis, DH). Furthermore, a non-copper associated subclinical hepatitis group (N-CASH) with normal hepatic copper concentrations was added as a control. Most mRNA levels of proteins involved in copper binding, transport, and excretion were around control values in the N-CASH and CASH group. In contrast, many of these (including ATP7A, ATP7B, ceruloplasmin, and metallothionein) were significantly reduced in the DH group. Measurements on defences against oxidative stress showed a decrease in gene-expression of superoxide dismutase 1 and catalase in both groups with high copper. Moreover, the anti-oxidative glutathione molecule was clearly reduced in the DH group. CONCLUSION: In the DH group the expression of gene products involved in copper efflux was significantly reduced, which might explain the high hepatic copper levels in this disease. ROS defences were most likely impaired in the CASH and DH group. Overall, this study describes a new variant of primary copper toxicosis and could provide a molecular basis for equating future treatments in dog and in man.