The role of prokineticins in recurrent implantation failure.

The aim of the present study was to investigate the expression patterns of prokineticins (PROK) and prokineticin receptors (PROKR) in the endometrium of women with recurrent implantation failure (RIF). Fifteen (15) women with RIF and 15 fertile controls were enrolled in this study. Endometrial samples were taken from study participants with an endometrial biopsy cannula during the implantation window. Real time polymerase chain reaction and immunohistochemistry were used to determine PROK/PROKR mRNA expression and protein localization, respectively. PROK1 mRNA levels were 6.09 times higher compared to endometrial samples obtained from women with RIF than in samples obtained from fertile controls, whereas PROKR1 mRNA levels were 2.46 times lower in endometrial samples obtained from women with RIF than in samples from fertile controls. In addition, decreased PROKR1 was supported by immunohistochemistry analysis at protein level. There was no statistically significant difference between women with RIF and fertile controls regarding PROK2 and PROKR2 levels. Altered expression of the PROK1/PROKR1 system could be one of the numerous abnormalities in the endometrium of women with RIF.

Prokineticin 1 is a new biomarker of human oocyte competence: expression and hormonal regulation throughout late folliculogenesis.

CONTEXT: Prokineticin 1 (PROK1) quantification in global follicular fluid (FF) has been recently reported as a predictive biomarker of in vitro fertilization (IVF) outcome. It is now necessary to evaluate its clinical usefulness in individual follicles. OBJECTIVES: To evaluate the clinical value of PROK1 secretion in individual FF to predict oocyte competence. To determine the impact of follicular size, oocyte maturity, and gonadotropin treatments on PROK1 secretion. DESIGN AND SETTING: Prospective cohort study from May 2015 to May 2017 at the University Hospital of Grenoble. PATIENTS: A total of 69 infertile couples underwent IVF. INTERVENTION(S): Collection of 298 individual FF from 44 women undergoing IVF; 52 individual cumulus cell (CC) samples and 15 CC primary cultures from 25 women undergoing IVF-intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Oocyte competence was defined as the ability to sustain embryo development to the blastocyst stage. Follicular size was measured by 2D-sonography. PROK1 concentration was quantified by ELISA assay. RESULTS: PROK1 concentration was correlated to follicular size (r = 0.85, P = 2.2 × 10-16). Normalized PROK1 concentration in FF was predictive of subsequent oocyte competence (AUROC curve = 0.76 [95% CI, 0.69-0.83]; P = 1.7 × 10-9), irrespectively of day-2 embryo morphokinetic parameters. The expression and secretion of PROK1 were increased in FF and CC of mature oocytes (P < 0.01). Follicle Stimulating Hormone and hCG up-regulated PROK1 secretion in CC primary cultures (P < 0.01; P < 0.05), probably through the cAMP pathway (P < 0.01). CONCLUSIONS: PROK1 quantification in individual FF could constitute a new predictive biomarker of oocyte competence in addition with embryo morphokinetic parameters. TRIAL REGISTRATION NUMBER: none.

EG-VEGF Maintenance Over Early Gestation to Develop a Pregnancy-Induced Hypertensive Animal Model.

During the last decade, multiple animal models have been developed to mimic hallmarks of pregnancy-induced hypertension (PIH) diseases, which include gestational hypertension, preeclampsia (PE), or eclampsia. Converging in vitro, ex vivo, and clinical studies from our group strongly suggested the potential involvement of the new angiogenic factor EG-VEGF (endocrine gland-derived-VEGF) in the development of PIH. Here, we described the protocol that served to demonstrate that maintenance of EG-VEGF production over 11.5 days post coitus (dpc) in the gravid mice caused the development of PIH. The developed model exhibited most hallmarks of preeclampsia.

PROK1 Level in the Follicular Microenvironment: A New Noninvasive Predictive Biomarker of Embryo Implantation.

CONTEXT: Prokineticin 1 (PROK1), also called endocrine gland-derived vascular endothelial growth factor, is a well-established regulator of endometrial receptivity and placental development. However, its clinical usefulness as a noninvasive predictive biomarker of embryo implantation is yet to be validated. OBJECTIVE: The main objective of this article was to determine the relationship between PROK1 levels in the follicular fluid (FF) and fertilization culture media (FCM) and the reproductive outcome in patients who received a first conventional in vitro fertilization-embryo transfer. The secondary objective was to characterize the expression of PROK1 and its receptors (PROKRs) in the human follicular microenvironment. DESIGN AND SETTING: We conducted a prospective study between January 2013 and June 2015 at the University Hospital of Grenoble. PATIENTS: A total of 135 infertile in vitro fertilization patients and 10 women undergoing ovarian tissue cryopreservation were included. INTERVENTIONS: The PROK1 concentration was measured by ELISA in FF and FCM collected on the day of oocyte retrieval and the day of the oocyte denudation step, respectively. Follicular expression of the PROK1/PROKR system was determined by immunohistochemistry, RT-quantitative PCR, and ELISA. MAIN OUTCOME MEASURE: Assessment of the clinical pregnancy rates was the main outcome. RESULTS: FF and FCM PROK1 levels were significantly higher in the embryo implantation group (P < .001) and were predictive of subsequent embryo implantation (area under the receiver operating characteristic curve, 0.91 [95% confidence interval, 0.81-1.00], P = .001; and 0.88 [0.72-1.00], P = .001, respectively). FF and FCM PROK1 levels remain similar irrespective of the embryo morphokinetic parameters (P = .71 and P = .83, respectively). The PROK1/PROKR system is expressed during human folliculogenesis. CONCLUSIONS: PROK1 levels in FF and FCM could constitute new predictive noninvasive markers of successful embryo implantation in conventional in vitro fertilization-embryo transfer.

The prognosis was poorer in colorectal cancers that expressed both VEGF and PROK1 (No correlation coefficient between VEGF and PROK1).

The angiogenic proteins vascular endothelial growth factor (VEGF) and prokineticin1 (PROK1) proteins are considered important in colorectal cancer, the relationship between their simultaneous expression and prognosis was investigated in the present study. VEGF and PROK1 expression in 620 primary human colorectal cancer lesions was confirmed via immunohistochemical staining with anti-VEGF and anti-PROK1 antibodies, and the correlation between the expression of these 2 proteins and recurrence/prognosis were investigated. VEGF protein was expressed in 329 (53.1%) and PROK1 protein was expressed in 223 (36.0%). PROK1 and VEGF were simultaneously expressed in 116 (18.7%) of the 620 cases. The correlation coefficient between VEGF expression and PROK1 expression was r = 0.11, and therefore correlation was not observed. Clinical pathology revealed that substantially lymphnode matastasis, hematogenous metastasis, or TMN advanced-stage IV was significantly more prevalent in cases that expressed both VEGF and PROK1 than in the cases negative for both proteins or those positive for only 1 of the proteins. Also the cases positive for both proteins exhibited the worst recurrence and prognosis. In the Cox proportional hazards model, VEGF and PROK1 expression was an independent prognostic factor. The prognosis was poorer in colorectal cancers that expressed both PROK1 and VEGF relative to the cases that expressed only 1 protein, and the expression of both proteins was found to be an independent prognostic factor.

Anti-prokineticin1 (PROK1) monoclonal antibody suppresses angiogenesis and tumor growth in colorectal cancer.

BACKGROUND: The prokineticin1 (PROK1) gene has been cloned as an angiogenic growth factor from endocrine gland cells. However, we have not known about potentials of anti-PROK1 monoclonal antibody in human cancers. Here we investigated how the anti-PROK1 monoclonal antibody (mAb; established by our department) would affect the high-PROK1-expressing colorectal cancer (CRC) cells in vitro and vivo. METHODS: We confirmed PROK1 protein expression in the CRC cells by performing immunohistochemical staining and measured the amount of soluble PROK1 protein. Next, we mixed the CRC cell culture fluid with the anti-PROK1mAb to examine angiogenic activity in vitro and in vivo. Additionally, we investigated whether the anti-PROK1mAb would affect the tumor-forming capability of high PROK1-expressing CRC cells implanted into mice. RESULTS: PROK1 protein expression was confirmed in 3 CRC cell lines, and soluble PROK1 protein was also confirmed in the CRC cell culture fluid. The culture fluid increased angiogenesis in vitro and vivo, whereas the anti-PROK1mAb suppressed angiogenesis. Subcutaneous tumor formation and tumor angiogenesis in mice were suppressed by the anti-PROK1mAb treatment. The anti-PROK1mAb significantly suppressed the number of CD31 stained cells in mice. CONCLUSIONS: The in vitro and vivo experimental system indicated that the anti-PROK1mAb could suppress angiogenesis and tumor growth in the CRC strains.

[PROK1, prognostic marker of embryo implantation?]. / PROK1, biomarqueur de l'implantation embryonnaire ?

In spite of improvements in assisted reproductive technology (ART) during the last 30 years, the rate of pregnancy remains constrained, as only about 25 % of embryo transfer lead to successful pregnancies, even with an average of two embryos replaced. Embryo selection is currently based on the establishment of morphokinetic scores, a method that obviously exhibits limitations. Therefore, the assessment of embryo development potency by criteria of higher predictive value is mandatory in order to increase the rates of pregnancy. Nowadays, there is increasing evidence that angiogenic factors might contribute to the success of the implantation and to the pregnancy outcome. Among these factors, prokineticin 1 (PROK1) and its receptors (PROKRs) constitute new targets that showed over the last ten years strong biological features directly linked to ovarian physiology, endometrial receptivity, embryo implantation and thus successful pregnancies. In ART, the rates of circulating PROK1 were reported in 2012 as significantly linked to the quality of embryonic cohort, as well as to the rates of pregnancy. Our preliminary data suggest a high potential of this cytokine in the success of implantation and pregnancy, and strongly overtones the emergency to investigate the value of its measurement in conditioned media of oocytes and embryo cultures in ART.

Effects of EG-VEGF, VEGF and TGF-ß1 on pregnancy outcome in patients undergoing IVF-ET treatment.

PURPOSE: To investigate the correlation of endocrine gland-derived vascular endothelial growth factor (EG-VEGF), vascular endothelial growth factor (VEGF) and transforming growth factor beta 1 (TGF-ß1) with the corresponding reproductive outcome in patients who received in vitro fertilization-embryo transfer (IVF-ET). METHODS: Sixty-seven women undergoing IVF-ET at a university tertiary hospital were recruited for a prospective study. Concentrations of EG-VEGF, VEGF and TGF-ß1 were measured by enzyme-linked immunosorbent assay (ELISA) in follicular fluid (FF) collected during oocyte retrieval (OR) and in serum collected 2 days after OR. RESULTS: In FF, concentrations of both EG-VEGF and VEGF were negatively correlated with peak E2 and the number of MII oocytes retrieved, and positively correlated with each other. In serum, concentrations of all the three growth factors were positively correlated with the rate of good quality embryo, and with one another. Patients in the pregnancy group had lower peak E2 concentrations and higher serum EG-VEGF concentrations than those in the non-pregnancy group, but such tendency was not observed in the case of VEGF and TGF-ß1. CONCLUSIONS: Both concentrations of EG-VEGF and VEGF in FF were negatively correlated with ovarian response and oocyte maturation. Concentrations of all the three growth factors in serum were positively correlated with embryo quality, but only serum concentrations of EG-VEGF were associated with the pregnancy outcome.

Potential biomarkers of temporomandibular joint disorders.

PURPOSE: The purpose of this study was to identify protein markers present in subjects with temporomandibular joint disorders (TMDs) and clicking compared with the levels in controls. MATERIALS AND METHODS: This was a pilot case-control study, and we report the preliminary results. Samples of joint aspirate collected from patients with TMDs and controls who had undergone surgery for a problem other than TMDs were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and biotin-labeled-based protein arrays. The data obtained from these techniques were used to identify the proteins of interest, which were then quantitated using enzyme-linked immunosorbent assay (ELISA). The patient samples studied included joint aspirate collected clinically from the controls and patients and included samples from both the right and the left sides of each patient with a TMD. RESULTS: The 8 TMJ aspirate samples from 6 subjects included 5 aspirate samples from 4 patients and 3 from 2 controls. The greatest standardized protein concentration of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/PK1) and D6 was found in both joints of the controls compared with the levels from the joints of the patients. With 1 exception, the standardized protein concentration was significantly lower in the patients than in the controls. The lower levels of EG-VEGF/PK1 and D6 in the patients compared with the controls suggest that these cytokines might be possible biomarkers for TMDs. CONCLUSION: In the present pilot study, greater levels of EG-VEGF/PK1 and D6 were found in the controls than in the patients with TMDs. Proteomic analysis of the proteins present in the diseased joints compared with those in the controls might help to identify proteins present when pain or degeneration of the joint occurs. The proteomic information might be useful in the development of future therapies.

Endocrine gland-derived vascular endothelial growth factor concentrations in follicular fluid and serum may predict ovarian hyperstimulation syndrome in women undergoing controlled ovarian hyperstimulation.

OBJECTIVE: To assess the predictive value of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) concentrations in follicular fluid (FF) and serum for ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation. DESIGN: Retrospective, case-control study. SETTING: University hospital, IVF center. PATIENT(S): Seventeen women with OHSS and 61 controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): FF and serum EG-VEGF and VEGF concentrations, IVF outcome. RESULT(S): FF and serum EG-VEGF concentrations showed a significant negative correlation with serum E(2) concentration on the day of hCG administration. FF, but not serum, VEGF concentrations also showed a significant negative correlation with serum E(2) concentrations on hCG day. The FF EG-VEGF, FF VEGF, and serum EG-VEFG concentrations were significantly lower in the OHSS group than in the non-OHSS group. There was no significant difference in serum VEGF concentrations. Among FF and serum EG-VEGF and VEGF concentrations, only FF EG-VEGF concentrations were significantly lower in patients with moderate OHSS than in those with mild OHSS. CONCLUSION(S): FF and serum EG-VEGF concentrations may predict OHSS occurrence. Furthermore, FF EG-VEGF concentrations were significantly correlated with OHSS severity; thus, EG-VEGF appears to be more valuable than VEGF for predicting OHSS.

Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered "superfertile", defined by a mean TTP of 3 months or less. CONCLUSIONS: Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor.

Implantation requires communication between a receptive endometrium and a healthy blastocyst. This maternal-embryonic crosstalk involves local mediators within the uterine microenvironment. We demonstrate that a secreted protein, prokineticin 1 (PROK1), is expressed in the receptive endometrium and during early pregnancy. PROK1 induces expression of leukemia inhibitory factor (LIF) in endometrial epithelial cells and first trimester decidua via a Gq-Ca(2+)-cSrc-mitogen-activated protein kinase kinase-mediated pathway. We show that human embryonic chorionic gonadotropin (hCG) induces sequential mRNA expression of PROK1 and LIF in an in vivo baboon model, in human endometrial epithelial cells, and in first-trimester decidua. We have used micro RNA constructs targeted to PROK1 to demonstrate that hCG-mediated LIF expression in the endometrium is dependent on prior induction of PROK1. Dual immunohistochemical analysis colocalized expression of the luteinizing hormone/chorionic gonadotropin receptor, PROK1, PROKR1, and LIF to the glandular epithelial cells of the first trimester decidual tissue. PROK1 enhances adhesion of trophoblast cells to fibronectin and laminin matrices, which are mediated predominantly via LIF induction. These data describe a novel signaling pathway mediating maternal-embryonic crosstalk, in which embryonic hCG via endometrial PROK1 may play a pivotal role in enhancing receptivity and maintaining early pregnancy.

The endocrine-gland-derived vascular endothelial growth factor (EG-VEGF)/prokineticin 1 and 2 and receptor expression in human prostate: Up-regulation of EG-VEGF/prokineticin 1 with malignancy.

A new family of angiogenic factors named endocrine-gland-derived vascular endothelial growth factors (EG-VEGF)/prokineticins (PK) have been recently described as predominantly expressed in steroidogenic tissues. Whether the normal and malignant epithelial prostate cells and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. We studied the expression of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in human prostate and their involvement in cancer. Using immunohistochemistry, Western blot, and RT-PCR, we determined the expression of EG-VEGF/PK1 in normal prostate (NP) and malignant prostate tissues (PCa), in epithelial cell primary cultures from normal prostate (NPEC) and malignant prostate (CPEC) and in a panel of prostate cell lines. In NPEC, CPEC, and in EPN, a nontransformed human prostate epithelial cell line, EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels were evaluated by quantitative RT-PCR. EG-VEGF/PK1 transcript was found in PCa, in CPEC, in EPN, and in LNCaP, whereas it was detected at low level in NP and in NPEC. EG-VEGF/PK1 was absent in androgen-independent PC3 and DU-145 cell lines. Immunochemistry confirmed that EG-VEGF/PK1 protein expression was restricted to hyperplastic and malignant prostate tissues, localized in the glandular epithelial cells, and progressively increased with the prostate cancer Gleason score advancement. EG-VEGF/PK1 and PK2 were weakly expressed in NPEC and EPN. On the other hand, their transcripts were highly detected in CPEC. PK-R1 and PK-R2 were found in NPEC, EPN, and CPEC. Interestingly, CPEC showed a significantly (P < 0.05) higher expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 compared with NPEC and EPN. We demonstrated that PKs and their receptors are expressed in human prostate and that their levels increased with prostate malignancy. It may imply that EG-VEGF/PK1 could be involved in prostate carcinogenesis, probably regulating angiogenesis. Thus, the level of EG-VEGF/PK1 could be useful for prostate cancer outcome evaluation and as a target for prostate cancer treatment in the future.

Expression and oxygen regulation of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 and its receptors in human placenta during early pregnancy.

Angiogenesis is a key process of dynamic tissue remodeling occurring during placentation. Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be deregulated in preeclampsia. Recently a new angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), also known as prokineticin 1 (PK1), has been identified, and its expression was shown to be restricted to endocrine glands, including the placenta. In this study we investigated the pattern of expression of EG-VEGF, its related factor Bv8/PK2, and their common receptors, PKR1 and PKR2, in human placenta during the first trimester of pregnancy. We also examined EG-VEGF and PKR1 regulation by oxygen tension in isolated trophoblast cells (TCs). Our results show that EG-VEGF, but not Bv8/PK2, is expressed in human placenta. EG-VEGF is mainly localized to the syncytiotrophoblast layer with the highest expression detected between the 8th and 10th wk of gestation. EG-VEGF expression within placental villi is different from that of VEGF, which is mainly localized in the cytotrophoblast and extravillous trophoblast cells. In TCs, PKR1 mRNA is about 80 times more abundant than PKR2 mRNA. Both EG-VEGF and PKR1 mRNAs appear to be regulated by hypoxia. These findings suggest that EG-VEGF has a direct effect on TCs via its receptor PKR1 and is likely to play an important role in human placentation. The expression pattern of EG-VEGF, its regulation by oxygen tension, and its complementary localization to that of VEGF suggest that this new factor might also be deregulated in preeclampsia.