Differences in secretome in culture media when comparing blastocysts and arrested embryos using multiplex proximity assay.

OBJECTIVES: The aim of this study was to assess different patterns of the human embryo secretome analysed as protein levels in culture media. Furthermore, analyses to correlate protein levels with quality and timing to development of human embryos were performed. MATERIAL AND METHODS: Human day-2 cryopreserved embryos were cultured for four days in an EmbryoScope® with a time-lapse camera, and embryo quality was evaluated retrospectively. After culture, the media were collected and relative levels of secreted proteins were analysed using Proseek Multiplex Assays. Protein levels were evaluated in relation to timing to development and the ability to form a blastocyst. RESULTS: Specific patterns of timing of development of blastocysts were found, where a difference in time to start of cavitation was found between high- and low-quality blastocysts. There appeared to be a correlation between specific protein patterns and successful formation of morulae and blastocysts. Embryos developing into blastocysts had higher levels of EMMPRIN than arrested embryos, and levels of caspase-3 were lower in high- versus low-quality blastocysts. Also, higher levels of VEGF-A, IL-6, and EMMPRIN correlated with shorter times to morula formation. CONCLUSIONS: The secretome and timing to development differ in embryos forming blastocysts and those that become arrested, and in high- versus low-quality blastocysts. The levels of certain proteins also correlate to specific times to development.

Relationship of DNA integrity to HRG C633T SNP and ART outcome in infertile couples.

The status of sperm DNA fragmentation, protamine deficiency, free thiols and disulphide bonds in colloid-selected samples and its relationship to ART outcome or HRG C633T SNP is not known. The objective of this study was to determine these relationships in spermatozoa from men with male factor or unknown factor infertility (n = 118) undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Sperm DNA integrity was analysed by flow cytometry using three fluorescent probes (acridine orange, monobromobimane and chromomycin A3). Principal component analysis (PCA) was used to identify the parameters that most influenced fertility. The relationships of sperm DNA integrity with seminal parameters, HRG C633T SNP and ART outcome were established using ANOVA and t-test. Sperm concentration and yield after preparation accounted for 27% of the total variance; sperm DNA integrity (%DFI and disulphide bonds) accounted for 16% of the variance in men from infertile couples. Sperm %DFI was significantly higher (P < 0.05) in older men than in younger men. A significant difference (P < 0.01) was observed in %DFI between smokers and non-smokers. Sperm %DFI was significantly higher (P < 0.01) in male factor infertility compared to either female factor or unknown factor infertility while free thiols were significantly higher (P < 0.01) in unknown infertility factor. No significant difference was observed between IVF success/failure in any of the seminal parameters studied. There was a tendency for protamine deficiency to be higher and disulphide concentration to be lower in men with HRG 633T. Such assessments may provide additional useful information about the prognosis for ART outcome, although more research is needed before clinical guidelines can be provided.

The effect of a specific histidine-rich glycoprotein polymorphism on male infertility and semen parameters.

In women, there is evidence that a single nucleotide polymorphism (SNP) in the histidine-rich glycoprotein (HRG) named HRG C633T is relevant for a number of fertility outcomes including recurrent miscarriage, ovarian response and pregnancy outcome after IVF. This case-control study was designed to investigate whether the HRG C633T SNP is important for male infertility and pregnancy rate following IVF. Cases were 139 infertile couples and controls were 196 pregnant couples. The 335 couples all contributed with one blood sample per partner. Genomic DNA was extracted and genotyping was performed using a TaqMan® SNP Genotyping Assay. Information on pregnancy rate and semen parameters was derived from medical records. Infertile couples in which the male partner was a homozygous carrier of the HRG C633T SNP had significantly lower (P < 0.01) pregnancy rate following IVF in comparison with couples where the male partner was a heterozygous HRG C633T SNP carrier. Male homozygous HRG 633T SNP carriers had overall lower total sperm count, sperm concentration, motility score and yield after preparation. In conclusion, once infertility is established the HRG C633T SNP seems to be important for male infertility and pregnancy rate following IVF.

Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development.

UNLABELLED: Histidine-rich glycoprotein (HRG) is an abundant plasma protein involved in multiple biological processes including immunology, vascularisation, and coagulation. These processes are of importance in regulating embryo development and implantation. A specific polymorphism in the HRG gene, HRG C633T, has an impact on various aspects of fertility, such as oocyte quality, endometrial receptivity, and possibly the capacity of the embryo itself to implant. To further examine the potential role of the HRG C633T polymorphism in regulating endometrial angiogenesis and on embryo development, two HRG peptides were constructed. These HRG peptides correspond to the amino acids 169-203 of the protein which, in turn, reflects the C633T polymorphism in the gene. The HRG proline or serine peptides were added to cultures of primary human endometrial endothelial (HEE) cells and to human embryos in vitro. The HRG peptides inhibited vascular endothelial growth factor (VEGF) induced proliferation and migration and promoted tube formation of HEE cells. The embryos were monitored using a time-lapse system (EmbryoScope®). Except for a prolonged time from first cleavage after thawing to development of the morula, no difference in embryo morphokinetics or embryo quality was noted in human embryos cultured in the presence of the HRG proline peptide. Taken together, these results suggest that treatment with a specific HRG peptide might prime the endometrium for implantation and be beneficial for adequate placentation. However, addition of a specific HRG proline peptide to human embryos has no beneficial effects in terms of embryo development. ABBREVIATIONS: HRG: histidine-rich glycoprotein; HEE: human endometrial endothelial; VEGF: vascular endothelial growth factor; TSP: thrombospondin; SNP; single nucleotide polymorphism; IVF: in vitro fertilization; CLESH-1: CD36 LIMPII Emp structural homology domain-1; ECM: endothelial cell medium; FBS: fetal bovine serum; cDNA: complementary DNA.

Ovarian response is affected by a specific histidine-rich glycoprotein polymorphism: a preliminary study.

Genetic polymorphisms involved in angiogenesis, apoptosis and chemokine signalling are associated with varying ovarian response and oocyte quality. The protein, histidine-rich glycoprotein (HRG), is involved in these processes, but its effect on ovarian response in IVF has not been previously studied. A single nucleotide polymorphism (SNP) in the HRG gene (C633T) seems to affect pregnancy results in IVF. Women with the C/C genotype had higher pregnancy rates, C/T had moderate rates and none of those in the T/T group conceived. The aim of this study was to investigate if the HRG C633T SNP affects ovarian response. The HRG C633T SNP genotype of 67 women with unexplained infertility undergoing IVF was analysed and related to medical data. The T/T genotype obtained fewer oocytes, including mature oocytes, despite higher dosages of FSH administered. Additionally, the highest proportion of women who had exclusively poor-quality embryos was in the T/T group. No differences in demographic factors known to affect these parameters were found. The results suggest that the HRG C633T SNP influences ovarian response. Further studies of this SNP may increase knowledge about the biological processes involved in oocyte development and, furthermore, improve predicted ovarian response and fertilization.

Histidine-rich glycoprotein gene polymorphism in patients with recurrent miscarriage.

Association between the histidine-rich glycoprotein (HRG) C633T single nucleotide polymorphism (SNP) and recurrent miscarriage was investigated in a case-control study. The cases constituted 187 women with recurrent miscarriage that were compared with 395 controls who had delivered a child and had no history of miscarriage. Blood samples were collected from each woman, genomic DNA was extracted and genotyped for the HRG C633T SNP. In the whole study population, the percentage of miscarriage was the same, regardless of genotype (C/C 31.2%, C/T 32.9% and T/T 32.5%). However, an association between homozygous T/T carriers and recurrent miscarriage was detected in a subgroup of women with primary recurrent miscarriage (odds ratio 2.44, 95% CI 1.01-5.92). Our results indicate an important role for the HRG C633T SNP in the occurrence of recurrent miscarriage.

Histidine-rich glycoprotein polymorphism and pregnancy outcome: a pilot study.

Histidine-rich glycoprotein (HRG) is involved in fibrinolysis and coagulation, the immune system and angiogenesis. These processes are all crucial in establishing and maintaining pregnancy. The primary aim of this pilot study was to determine if HRG affects pregnancy outcome. The secondary aim was to investigate if a specific genetic polymorphism (rs9898 C/T) in the HRG gene is associated with pregnancy results. The polymorphism leads to expression of either a serine or proline residue at position 186 in the protein sequence. In this study, women undergoing IVF were included. The genetic polymorphism in the HRG gene was analysed by Western blot and single nucleotide polymorphism analysis. None of the women homozygous for the serine at residue 186 became pregnant whereas the women homozygous for proline at residue 186 had higher than expected pregnancy rates. As far as is known,this is the first study to show that a specific genetic polymorphism in the HRG gene of a woman affects her chances of becoming pregnant after IVF. The results may be essential in improving advice and IVF treatment for couples with unexplained infertility.

The presence of histidine-rich glycoprotein in the female reproductive tract and in embryos.

A well-regulated angiogenesis is crucial for proper embryo implantation, embryogenesis, and pregnancy development. Monitoring the presence and distribution of angiogenic regulators in the female reproductive tract and in the early embryo is important for a broader understanding of the molecular aspects of fertility, embryogenesis, and pregnancy. Histidine-rich glycoprotein (HRG) is a glycoprotein involved in angiogenesis. Its presence in the female reproductive tract or in embryos has not previously been studied. Follicular fluid, culture medium, and embryos were obtained from patients undergoing in vitro fertilization (IVF). Biopsies from inner genitalia and placenta were collected at surgery. Histidine-rich glycoprotein presence was investigated by immunohistochemistry and Western blot. Polymerase chain reaction (PCR) was used to determine HRG expression in tissues or by embryos. We identified HRG in follicular fluid, the female reproductive tract, and placenta, as well as in the embryos. Moreover, HRG expression was observed in blastocysts. Thus, the angiogenic properties of HRG might affect fertility.

Fibrinogen and histidine-rich glycoprotein in early-onset preeclampsia.

OBJECTIVE: To determine whether plasma levels of fibrinogen and the placental tissue distributions of fibrinogen and histidine-rich glycoprotein (HRG) differ between early- and late-onset preeclampsia. DESIGN: The study comprised 18 women with early-onset (gestational weeks 24-32) and 19 women with late-onset (gestational weeks 35-42) preeclampsia. As controls concerning the plasma levels of fibrinogen, we used samples from non-pregnant fertile women, healthy pregnant women at gestational weeks 24-32 and healthy pregnant women at gestational weeks 35-42. Placental samples from women with healthy pregnancies at gestational weeks 35-42 served as controls in the immunohistochemical staining. SETTING: Uppsala University Hospital, Uppsala. METHODS: Plasma fibrinogen levels were analyzed and the placental tissue expression of fibrinogen and HRG determined by immunohistochemistry. RESULTS: Plasma level of fibrinogen was increased in early-onset, but not late-onset, preeclampsia. Levels of fibrinogen were significantly lower, and that of HRG significantly higher, in placentas from women with early-onset preeclampsia as compared with control placentas (p = 0.01 and 0.001). CONCLUSIONS: HRG and fibrinogen might be involved in the hypercoagulability and the angiogenic imbalance seen in early-onset preeclampsia.

Activated platelets provide a functional microenvironment for the antiangiogenic fragment of histidine-rich glycoprotein.

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine- and proline-rich (His/Pro-rich) domain. Using a combination of immunohistochemistry and mass spectrometry, we here provide biochemical evidence for the presence of a proteolytic peptide, corresponding to the antiangiogenic domain of HRG, in vivo in human tissue. This finding supports a role for HRG as an endogenous regulator of angiogenesis. Interestingly, the His/Pro-rich peptide bound to the vessel wall in tissue from cancer patients but not to the vasculature in tissue from healthy persons. Moreover, the His/Pro-rich peptide was found in close association with platelets. Relesate from in vitro-activated platelets promoted binding of the His/Pro-rich domain of HRG to endothelial cells, an effect mediated by Zn(2+). Previous studies have shown that zinc-dependent binding of the His/Pro-rich domain of HRG to heparan sulfate on endothelial cells is required for inhibition of angiogenesis. We describe a novel mechanism to increase the local concentration and activity of an angiogenesis inhibitor, which may reflect a host response to counteract angiogenesis during pathologic conditions. Our finding that tumor angiogenesis is elevated in HRG-deficient mice supports this conclusion.

IFN-gamma-induced upregulation of Fcgamma-receptor-I during activation of monocytic cells requires the PKR and NFkappaB pathways.

Interferon (IFN)-gamma is a potent activator of macrophages, increasing the cells capacity to perform specific functions during inflammation and immune response. In this report we use IFN-gamma-induced upregulation of the high affinity receptor for IgG (FcgammaRI/CD64) in the human monocytic cell line U-937 as a model for monocytic activation. We show that upregulation of FcgammaRI is dependent on signals mediated by the dsRNA-dependent kinase PKR, and the transcription factor NFkappaB. Silencing of PKR expression by siRNA or inhibition of PKR by 2-aminopurine (2-AP) potently blocks the IFN-gamma-induced transcriptional activation of the FcgammaRI promoter. We find that the serine 727 phosphorylation of Stat1, required for full IFN-gamma-induced FcgammaRI promoter activity, is dependent on PKR. We further show that IFN-gamma induction of FcgammaRI upregulation is dependent on the NFkappaB pathway, as evidenced by inhibition of NFkappaB using a phosphorylation defective IkappaBalpha (S32A/S36A) mutant, or inhibiting the IkappaB-kinase (IKK) by treatment with BMS345541. Our results suggest that IFN-gamma-induced increase of FcgammaRI expression requires the integration of two signalling events: PKR-dependent Stat1 serine 727 phosphorylation, and activation of NFkappaB.