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Size and spatial orientation of uterine tissue transplants on the peritoneum crucially determine the growth and cyst formation of endometriosis-like lesions in mice

Posted: October 11, 2010 at 10:13 am

BACKGROUND

In many studies in rodents, intraperitoneal endometriosis-like lesions are surgically induced by syngeneic or autologous transplantation of uterine tissue samples, which are sutured to the abdominal wall. However, until now the surgical techniques have not been standardized, and we address this issue here.

METHODS

Uterine tissue samples were transplanted to the peritoneum of C57BL/6 mice (four study groups, n = 7 each). Using non-invasive high-resolution ultrasound imaging over a period of 4 weeks, we analyzed growth characteristics and cyst formation of the endometriosis-like lesions which developed, in relation to mode of transplantation (syngeneic versus autologous), type of tissue fixed adjacent to the peritoneum (endometrium versus perimetrium), and size of tissue transplanted (2 versus 3 mm). Immunohistochemical analysis was also performed.

RESULTS

When the perimetrium, with underlying myometrium, was sutured next to the host peritoneum the endometriosis-like lesions which developed exhibited a higher growth rate (P< 0.05 versus endometrium), and contained more proliferating cell nuclear antigen (PCNA)-positive cells and an increased microvessel density (both P< 0.05 versus endometrium). In the group with 3 mm uterine tissue grafts, lesion growth was significantly decreased when compared with 2 mm samples (P< 0.05). However, the larger grafts developed more cysts throughout the observation period than the smaller ones. There was no difference between syngeneic and autologous endometriosis-like lesions.

CONCLUSIONS

Our study demonstrates that size and spatial orientation of peritoneally fixed uterine tissue samples crucially determine growth and cyst formation of endometriotic lesions in mice. These findings should improve the standardization and reliability of future studies, performed in the frequently used mouse model of surgically induced endometriosis.

Recommendation and review posted by G. Smith

In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility

Posted: October 11, 2010 at 10:13 am

BACKGROUND

Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential.

METHODS

In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 µg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section.

RESULTS

In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age.

CONCLUSIONS

NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Recommendation and review posted by G. Smith

Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor, amphiregulin

Posted: October 11, 2010 at 10:13 am

BACKGROUND

The LH surge promotes ovulation via activation of multiple signaling networks in the ovarian follicle. Studies in animal models have shown the importance of LH-induced activation of the epidermal growth factor (EGF)signaling network in critical peri-ovulatory events. We investigated the biological significance of regulatory mechanisms mediated by EGF-like growth factors during LH stimulation in humans.

METHODS

We characterized the EGF signaling network in mature human ovarian follicles using in vivo and in vitro approaches. Amphiregulin (AREG) levels were measured in 119 follicular fluid (FF) samples from IVF/ICSI patients. Biological activity of human FF was assessed using in vitro oocyte maturation, cumulus expansion and cell mitogenic assays.

RESULTS

AREG is the most abundant EGF-like growth factor accumulating in the FF of mature follicles of hCG-stimulated patients. No AREG was detected before the LH surge or before hCG stimulation of granulosa cells in vitro, demonstrating that the accumulation of AREG requires gonadotrophin stimulation. Epiregulin and betacellulin mRNA were detected in both human mural and cumulus granulosa cells, although at significantly lower levels than AREG. FF from stimulated follicles causes cumulus expansion and oocyte maturation in a reconstitution assay. Immunodepletion of AREG abolishes the ability of FF to stimulate expansion (P < 0.0001) and oocyte maturation (P < 0.05), confirming the biological activity of AREG. Conversely, mitogenic activity of FF remained after depletion of AREG, indicating that other mitogens accumulate in FF. FF from follicles yielding an immature germinal vesicle oocyte or from an oocyte that develops into an aberrant embryo contains lower AREG levels than that from follicles yielding a healthy oocyte (P = 0.008).

CONCLUSIONS

EGF-like growth factors play a role in critical peri-ovulatory events in humans, and AREG accumulation is a useful marker of gonadotrophin stimulation and oocyte competence.

Recommendation and review posted by G. Smith

Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice

Posted: October 11, 2010 at 10:12 am

Background:
Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.
Methods:
Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying ?-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.
Results:
Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.
Conclusion:
Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.

Recommendation and review posted by G. Smith

Kif 6, Genetic Findings = Useful Medicine 1 in 1000 times

Posted: October 11, 2010 at 10:12 am

Way back in 2008 I mentioned an article, which I hoped would pan out. Or at least I hoped it would point the way to a model of PGx research which would be followed by pharma and alike to find associations to help us target the right medication for the right patient.

While the similar model followed through with Plavix, the initial study did not.

Which is why when the Berkeley Heart Lab guy came last week, I told him I would not be testing for Kif6. It had not been replicated in further GWAS.

A VAP cholesterol panel, a HsCRP, a family history and a blood pressure can help me predict risk much better.

The problem and backlash facing DTCG and DTMD genetic test purveyors is

the 'Ol "Your million dollar major study now rushed to market has just been refuted"

Yes this happens in biomedical science and in medicine ALL THE TIME.
Bed rest for MI anyone?
Low Dose Dopamine?
I could go on and on, but I won't

Put simply, the majority of the genome is NOT ready for clinical medicine or clinical decision making.

It won't be for 20 years.

That doesn't mean there aren't some things we can use.

1. BRCA1/2
2. MMR genes
3. CYP2C19
4. CYP2D6, sometimes.....
5. SCD genes
6. Counsyl universal carrier screening

If a gene test comes to market that purports disease risk it had better be studied for at least 5 years before it comes to market.

Post market surveillance did not protect all those patients on Statins, "just because" of Kif6 risk.

Get it? These tests can lead to incorrect medical decisions....
Which can lead to risk.
Yes, even the DTCG tests can fool doctors and patients.

The Sherpa Says: If 2008 was the year of the GWAS, will 2011 be the year of the overturned GWAS?

Recommendation and review posted by G. Smith

A Whole Month Off, for Good Reason!

Posted: October 11, 2010 at 10:12 am

I have a lot of friends and readers who have emailed me over the month.

The recurring comments: "Has the DTCG field run you off of your blog?"

The answer: "Uh No"

Why I have I stopped blogging so much? For multiple reasons.

1. DTCG has been put on the Radar of the FDA and Government. I have nothing more to say about that.
2. Journalists and Genomics aficionados have been correctly pointing out the hype behind some tests. Most notably lately with the ADHD stuff, which BTW should not have been put out there in the press....Maybe, I need to blog more.....
3. My practice and patients have really taken up my time. I am working to apply personalized medicine daily. Because that is what is needed. We need to show the public and the press how it is done on a daily basis.

I will be back soon, to dissect shotty science and poor clinical studies. But for now, our boots are on the ground and we are climbing the mountain....

See you soon!

Recommendation and review posted by G. Smith


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