Category Archives: Human Reproduction

STUDY QUESTION

What is the methylation status of the Nanog and Oct4 promoters in human gametes and ICSI embryos and is abnormal reprogramming of their methylation associated with developmental failure of ICSI embryos?

SUMMARY ANSWER

Developmental failure of human ICSI embryos is associated with high methylation of the Oct4 promoter.

WHAT IS KNOWN ALREADY

Nanog and Oct4 genes play critical roles in the establishment and maintenance of pluripotency during normal early embryonic development, and both are negatively regulated through the methylation of their promoters.

STUDY DESIGN, SIZE AND DURATION

We analysed the methylation profile of Nanog and Oct4 promoters in 5 control sperm from normally fertile men, 70 metaphase II oocytes, 21 4-cell control ICSI embryos, 7 control blastocysts and 45 ICSI embryos arrested at 2- to 8-cell stage following prolonged culture.

PARTICIPANTS, MATERIALS, SETTING AND METHODS

Embryos and gametes were donated for research by patients from the Department of Reproductive Medicine at the Hôpital Femme Mère Enfant (Bron, France) and the Clinique du Tonkin (Villeurbanne, France) after giving their informed consent.

MAIN RESULTS

For both promoters, high methylation was observed in sperm cells. Although, in general, the promoters were unmethylated in oocytes, the methylation of some alleles was observed, particularly in oocytes from women with known infertility. Both gene promoters were hypomethylated in control blastocyst ICM (inner cell mass) and in control 2–8-cells embryos obtained from 6 out of 8 couples. However, they appeared highly methylated in embryos obtained from the other two couples. In most arrested ICSI embryos, the Nanog promoter was unmethylated while the Oct4 promoter was highly methylated. High methylation of the Oct4 promoter was significantly more pronounced in embryos from couples where a male factor was the only known cause of infertility. When the embryos were heterozygous for a G/A single nucleotide polymorphism, both alleles could be methylated, each likely representing a paternally inherited or a maternally inherited copy.

LIMITATIONS AND REASONS FOR CAUTION

The study was done on a limited number of oocytes and embryos and the gametes of the couples were not available.

WIDER IMPLICATIONS OF THE FINDINGS

These results provide new insight regarding the roles of epigenetic abnormalities in early developmental failure in humans.

STUDY FUNDING/COMPETING INTEREST(S)

No external funding was obtained for this study. There was no competing interest.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

STUDY QUESTION

What is the potential physiopathological role of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in recurrent miscarriage (RM), characterized by at least three consecutive pregnancy losses.

SUMMARY ANSWER

The levels of serum TRAIL immediately after miscarriage in RM patients are significantly elevated with respect to that in first-trimester normal pregnant women, and recombinant TRAIL inhibits the adhesion and migration of HTR8 trophoblastic cells in vitro.

WHAT IS KNOWN ALREADY

Both TRAIL and its trans-membrane receptors (TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4) have been documented in the placenta, but their physiopathological role is incompletely understood.

STUDY DESIGN, SIZE, DURATION

The study populations consisted of RM patients (n = 80) and first-trimester normal pregnant women (n = 80). Blood samples were obtained within 24 h after abortion (RM) or at gestational 12-week (normal pregnant women). As additional controls, third-trimester normal pregnant women (n = 28) were examined before (within 72 h) and after (within 24 h) partum.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The concentrations of TRAIL were analysed in serum samples by ELISA. In parallel, the effect of soluble recombinant TRAIL (0.1–1000 ng/ml) was analysed on the survival of primary extravillus trophoblasts (EVTs) and on the survival, proliferation, adhesion and migration of trophoblastic HTR8 cells.

MAIN RESULTS AND THE ROLE OF CHANCE

The circulating levels of TRAIL in RM women (median: 52.5 pg/ml; mean and SD: 55.5 ± 24.4 pg/ml) were significantly higher with respect to first-trimester normal pregnant women (median: 44.9 pg/ml; mean and SD: 47 ± 15.1 pg/ml) and third-trimester normal pregnant women, as assessed before (median: 45.1 pg/ml; mean and SD: 46 ± 12.4 pg/ml) and after partum (median: 35.4 pg/ml; mean and SD: 38 + 17.5 pg/ml). Both primary EVT and HTR8 cells expressed detectable levels of TRAIL death receptors, but exposure to soluble recombinant TRAIL did not induce cell death of trophoblastic cells. On the other hand, TRAIL dose-dependently inhibited the adhesion of HTR8 cells to decidual endothelial cells (DEC) as well as the migration of HTR8 in transwell assays using either fibronectin or DEC.

LIMITATIONS, REASONS FOR CAUTION

Although this study suggests that TRAIL might have a pathogenic role in RM by inhibiting both the adhesion and migration capabilities of first trimester trophoblastic cells, there is a possibility that the elevated serum levels of TRAIL in RM are not cause but rather the result of RM.

WIDER IMPLICATIONS OF THE FINDINGS

Our current findings together with data of other authors suggest that circulating TRAIL should be further analysed as a potential important biomarker in different physiopathological settings.

STUDY FUNDING/COMPETING INTEREST(S)

This study was funded by FIRB projects (RBAP11Z4Z9_002 to Giorgio Zauli and RBAP10447J_002 to Paola Secchiero). The authors have no competing interests to declare.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

STUDY QUESTION

What is the effect of pravastatin on antiphospholipid antibody (aPL) modulation of human first trimester trophoblast function?

SUMMARY ANSWER

Pravastatin does not prevent the effects of aPL on human first trimester trophoblast cell function.

WHAT IS KNOWN ALREADY

Antiphospholipid syndrome (APS) is associated with recurrent pregnancy loss and late pregnancy complications, such as pre-eclampsia, owing to direct targeting of the placenta by aPL. While treatment with heparin reduces the rate of pregnancy loss, the risk for severe pre-eclampsia remains high. Thus, there is a need to find alternative treatments for the prenatal management of patients with APS. Statins have recently been shown to prevent aPL-mediated fetal loss in mice but their effects on a human pregnancy model of APS have not yet been studied.

DESIGN, DATA COLLECTION, METHODS

The human first trimester trophoblast cell line, HTR8, and human first trimester trophoblast primary cultures were incubated with or without a mouse anti-human beta 2 glycoprotein I (β₂GPI) monoclonal antibody in the presence or absence of pravastatin. Cytokine and angiogenic factor secretion were measured by enzyme-linked immunosorbent assay and multiplex analysis. Cell migration was measured using a colorimetric two-chamber migration assay.

MAIN FINDINGS

Using the human first trimester trophoblast cell line, HTR8, pravastatin significantly augmented, compared with no treatment, aPL-dependent secretion of interleukin (IL)-8 (P< 0.05), IL-1β (P< 0.05) and soluble endoglin (P< 0.01) but had no effect on aPL-induced up-regulation of vascular endothelial growth factor, placenta growth factor or growth-related oncogene alpha secretion. Furthermore, pravastatin alone limited basal HTR8 cell migration (P< 0.01), and did not mitigate the adverse effect of aPL on trophoblast migration. Pravastatin also had no impact on the secretion of pro-inflammatory cytokines and angiogenic factors by primary human first trimester trophoblast cells exposed to aPL.

LIMITATIONS AND WIDER IMPLICATIONS OF THE FINDINGS

While our in vitro findings suggest that pravastatin may not be effective in preventing pregnancy complications in patients with APS, the in vivo condition may be more complex, and thus, more studies are needed to determine the effectiveness of pravastatin in the prevention of aPL-associated pregnancy complications in humans.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by the American Heart Association.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

STUDY QUESTION

Which is the role of microRNAs (miRNAs) related to several angiogenesis regulators such as VEGF-A (Vascular endothelial growth factor-A) and TSP-1 (Thrombospondin-1) in endometrial cancer?

SUMMARY ANSWER

A dysregulated expression of miRNAs related to angiogenesis and an increase in the VEGF-A levels were observed in endometrial cancer in comparison with control. The different expression of miRNAs could modulate the expression of angiogenic and antiangiogenic factors, which may play an important role in the pathogenesis of endometrial cancer.

WHAT IS KNOWN ALREADY

Dysregulated miRNA expression has been previously evaluated in endometrial adenocarcinoma. To the best of our knowledge, there are no studies on the relationship between angiogenic factors and miRNAs in endometrial cancer.

STUDY DESIGN, SIZE, DURATION

Case–control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer.

PARTICIPANTS/MATERIALS, SETTING, METHODS

RNAs isolated from tissue samples were analyzed using the GeneChip miRNA 2.0 Array platform (Affymetrix). TaqMan qRT–PCR was used to assess the expression of the selected miRNAs related to angiogenesis (miR-15b, -16, -17-5p, -20a, -21, -125a, -200b, -210, -214*, -221, -222 and -424), and VEGF-A and TSP-1 mRNAs were assessed by qRT–PCR using SYBR Green. Protein levels were quantified by ELISAs.

MAIN RESULTS AND THE ROLE OF CHANCE

Compared with the miRNAs in the control endometrium, eight miRNAs (miR-15b, -17-5p, -20a, -125a, -214*, -221, -222 and -424) were significantly down-regulated and two miRNAs (miR-200b and -210) were significantly up-regulated in the cancerous endometrium. A significant increase in VEGF-A mRNA and protein expression and in TSP-1 protein levels (P <0.01) was observed in endometrial cancer. Moreover, significant inverse correlations between VEGF-A protein levels and miR-20a, -125a, -214*, -221, -222 and -424 were detected. In contrast, a positive correlation was observed between VEGF-A and miR-200b and -210. Furthermore, stage IB endometrial cancer was associated with a higher VEGF-A protein/mRNA ratio and lower miR-214*, -221 and -222 expression in comparison with stage IA.

LIMITATIONS, REASONS FOR CAUTION

Future functional studies (e.g. miRNA inhibition or ectopic overexpression) in cell culture models are needed to confirm the VEGF targeting by the miRNAs found in the present study.

WIDER IMPLICATIONS OF THE FINDINGS

The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by research grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, PI080185, PI0110091) and Red RECAVA (RD06/0014/0004), by Consellería de Sanidad (AP-141/11) and Consellería de Educación (PROMETEO/2011/027), Generalitat Valenciana, by Beca Fibrinolisis 2009 and Becario 2010, 2011 from Fundación Española de Trombosis y Hemostasia and by the Fundación Investigación Hospital La Fe, Spain. None of the authors have any conflicts of interest.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

STUDY QUESTION

Is thymic stromal lymphopoietin (TSLP) involved in the pathophysiology of endometriosis?

SUMMARY ANSWER

TSLP is up-regulated by interleukin (IL)-1β and may be involved in the development of endometriosis.

WHAT IS KNOWN ALREADY

Endometriosis is a chronic inflammatory disease in which the Th2 immune response is activated and has been suggested to promote the disease. TSLP is a master cytokine that drive Th2 immune response.

STUDY DESIGN, SIZE, DURATION

A laboratory study.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Primary cultures of endometrioma stromal cells (ESCs) were treated with IL-1β, a typical inflammatory cytokine associated with endometriosis. Gene expression of TSLP in ESCs and secretion of TSLP protein from ESCs were studied using quantitative PCR and a specific ELISA. Interferon (IFN), a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, were added to the culture to evaluate their effect on the IL-1β-induced secretion of TSLP. Inhibitors of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) were added to the culture to examine intracellular signals involved in IL-1β-induced TSLP secretion. The expression of TSLP in endometrioma tissue was examined by immunohistochemistry. The concentration of TSLP in the serum and peritoneal fluid (PF) of women with or without endometriosis was measured with a specific ELISA.

MAIN RESULTS AND THE ROLE OF CHANCE

IL-1β stimulated the expression of TSLP mRNA and secretion of TSLP protein from ESCs. IL-4 enhanced the IL-1β-induced TSLP secretion from ESCs, while IFN reduced it. Inhibitors of p42/44 MAPK, p38 MAPK and SAPK/JNK suppressed the IL-1β-induced secretion of TSLP from ESCs. Positive immunostaining of TSLP was observed in the stroma of endometrioma tissue. TSLP concentrations in the serum and PF were both higher in women with endometriosis compared with those without endometriosis.

LIMITATIONS, REASONS FOR CAUTION

The present study was only in vitro. The samples used for culture were endometrioma tissues, not including other types of endometriosis. Therefore, the present findings should be interpreted with caution.

WIDER IMPLICATIONS OF THE FINDINGS

This study provided new insights in the Th2 immune response-related mechanism in endometriosis.

STUDY FUNDING

This study is partly supported by grants from the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology. The authors have no conflicts of interest to declare.

Source:
http://humrep.oxfordjournals.org/rss/current.xml

NEW YORK, Sept. 12, 2012 /PRNewswire/ --BRCA1/2 gene mutations, widely associated with breast and ovarian cancer risks in women, are, in principle, lethal to human embryos, according to new research conducted by three teams of researchers from the Center for Human Reproduction (CHR) in New York City, the Medical University Vienna in Vienna, Austria, and the Medical University Graz, Graz, Austria. BRCA1/2-positive embryos will only survive when also carrying a specific FMR1 gene genotype.

In a paper just published in the prestigious online medical journal PLoS ONE(1), the researchers examined the distribution of FMR1 genotypes and sub-genotypes amongst women with BRCA1/2 mutations and in a control population of infertile women. Unexpectedly, almost all the 99 carriers of BRCA1/2 mutations demonstrated a specific FMR1 genotype, the so-called "low" FMR1 allele, defined by less than 26 CGG triple nucleotides. In contrast, over 300 controls presented with a normal distribution of FMR1 genotypes and sub-genotypes.

The authors note that the most likely explanation for such a skewed distribution of FMR1 in BRCA1/2 mutation carriers is embryo lethality of BRCA1/2 in humans; only embryos carrying the "low" FMR1 allele are "rescued" from this embryo-lethality.

"We were very surprised by these results," says David H. Barad, MD, MS, Director of Clinical ART and Senior Scientist at CHR, a senior author of the study. "Since approximately 25% of all women have low FMR1 genotypes, this observation, if confirmed, can greatly impact current cancer screening methods for BRCA1/2-associated cancers in women, and greatly reduce costs."

"These findings also potentially explain the long-unexplained 'BRCA-paradox,'" notes Norbert Gleicher, MD, Medical Director and Chief Scientist of CHR, and another senior author of the study. "BRCA-paradox" refers to the fact that BRCA1/2 mutations are anti-proliferative in embryonic tissue but proliferative in cancer tissues. Dr. Gleicher continues: "Confirmed, these findings could mean that 'low' FMR1 alleles desuppress the antiproliferative activity of BRCA1/2 in both tissues, in embryonic tissues allowing the embryo to survive, while in cancers having the negative effect of allowing cancer to proliferate. This, of course, could open major therapeutic options for improving embryo growth and inhibiting cancer growth."

An Appellate Court recently reaffirmed Myriad Genetics' BRCA1/2 patent. Because of unusually high testing costs for BRCA1/2(ca. $3,000), breast cancer screening and ovarian cancer screening are currently recommended only for women with strong family histories of breast and ovarian cancers. This study suggests the possibility that much less costly FMR1 testing may be able to, at least partially, replace BRCA1/2 testing as a primary screening test. The FMR1 test application utilized in this research is pending U.S. patents, and has been licensed to Women's Laboratory Corporation, LLC, NY, NY.

(1) Weghofer et al, BRCA1/2 mutations appear embryo-lethal unless rescued by low (CGG n<26) FMR1 sub-genotypes: Explanation for the "BRCA paradox"? PLoS ONE 2012; http://dx.plos.org/10.1371/journal.pone.0044753

Drs. Barad and Gleicher are available for further comments in New York City. Dr. Weghofer, Associate Professor of Obstetrics & Gynecology at Vienna University, Visiting Associate Scientist at CHR and another senior author of the paper, is available for further comments from Vienna, Austria.

Read this article:
BRCA1/2 Mutation and FMR1 Gene Study Has Potential Implications for Cancer Screening and Treatment