NMR spectroscopy of a single mammalian early stage embryo.

The resolving power, chemical sensitivity and non-invasive nature of NMR have made it an established technique for in vivo studies of large organisms both for research and clinical applications. NMR would clearly be beneficial for analysis of entities at the microscopic scale of about 1 nL (the nanoliter scale), typical of early development of mammalian embryos, microtissues and organoids: the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 µm) and sensitivity issues have prevented a widespread adoption of NMR. In this article we show how these limitations can be overcome to obtain NMR spectra of a mammalian embryo in its early stage. To achieve this we employ ultra-compact micro-chip technologies in combination with 3D-printed micro-structures. Such device is packaged for use as plug & play sensor and it shows sufficient sensitivity to resolve NMR signals from individual bovine pre-implantation embryos. The embryos in this study are obtained through In Vitro Fertilization (IVF) techniques, transported cryopreserved to the NMR laboratory, and measured shortly after thawing. In less than 1 h these spherical samples of just 130-190 µm produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectra vary from one sample to another despite their optical and morphological similarities, suggesting that the method can further develop into a non-invasive embryo assay for selection prior to embryo transfer.

Ultra-low Doses of Follicle Stimulating Hormone and Progesterone Attenuate the Severity of Polycystic Ovary Syndrome Features in a Hyperandrogenized Mouse Model.

BACKGROUND: Polycystic-ovary syndrome (PCOS) is a reproductive illness characterized by hyperandrogenism and anovulation. Using hyperandrogenized mice, it was demonstrated that the oral administration of incremental dose of follicle stimulating hormone (FSH) attenuated some of PCOS characteristics. This work aimed to study the effect of ultra-low doses of combined FSH and progesterone orally administered on PCOS murine model. Moreover, the effect of sequential kinetic activation of administered hormones was tested. METHODS: Thirty-two female mice were used as animal model (four groups of eight animals each). Mice were hyperandrogenized by injection of dehyidroepiandrosterone diluted in sesame oil. Control group received only oil. Simultaneously, each animal daily received per os an activated or a not-activated combination of FSH (0.44 pg) plus progesterone (0.44 pg) or saline solution as control. Serum testosterone, estradiol, progesterone and luteinizing hormone were analyzed as endocrine markers and a morphological study of antral follicle was conducted. Data were analyzed by one-way ANOVA, followed by multiple comparison test. The p<0.05 was considered significant. RESULTS: Dehyidroepiandrosterone treatment increased both estradiol and progesterone serum levels, besides testosterone, while reduced luteinizing hormone (p<0.05); histological examination revealed an increase of cystic follicles (p<0.05). Irrespective of activation, the combined FSH and progesterone treatments restored estradiol level (p>0.05 vs. control group) and reduced cystic signs in the follicles (p<0.05 vs. dehyidroepiandrosterone treatment). CONCLUSION: This study indicate that ultra-low doses of FSH and progesterone orally administrated can reduce the sternness of PCOS in the mouse model and open a route for the study of innovative approaches for PCOS treatment.

Effect of oral administration of low-dose follicle stimulating hormone on hyperandrogenized mice as a model of polycystic ovary syndrome.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is a widespread reproductive disorder characterized by a disruption of follicular growth and anovulatory infertility. In women with PCOS, follicular growth and ovulation can be induced by subcutaneous injections of low doses of follicle stimulating hormone (FSH). The aim of this study was to determine the effect of oral administration of recombinant human FSH (rhFSH) on follicle development in a PCOS murine model. Moreover, since it is unlikely that intact rhFSH is present into the circulation after oral administration, the biological activity of a peptide fragment, derived from the predicted enzymatic cleavage sites with the FSH molecule, was investigated in vitro on cumulus-enclosed oocytes (COCs). METHODS: Female peripubertal mice were injected with dehydroepiandrosterone (DHEA) diluted in sesame oil for 20 consecutive days and orally treated with a saline solution of rhFSH. A control group received only sesame oil and saline solution. At the end of treatments, blood was analyzed for hormone concentrations and ovaries were processed for morphological analysis. The presumptive bioactive peptide was added during in vitro maturation of bovine COCs and the effects on cumulus expansion and on maturation rate were evaluated. RESULTS: DHEA treatment increased serum levels of testosterone, estradiol and progesterone as well as the percentage of cystic follicles. Orally administered rhFSH restored estradiol level and reduced the percentage of cystic follicles. Despite these results indicating a reduction of the severity of PCOS in the mouse model, the presumptive bioactive peptide did not mimic the effect of rhFSH and failed to induce bovine cumulus expansion and oocyte maturation in vitro. CONCLUSIONS: Although further studies are needed, the present data supports the concept that orally administrated FSH could attenuate some of the characteristic of PCOS in the mouse model.