Riassunto analitico
The glycoprotein hormones Follicule-Stimulating Hormone (FSH), Luteinizing Hormone (LH) and human Chorionic Gonadotropin (hCG) are fundamental for the regulation of gonadal function and exert their biological function by directly binding to their specific receptor, i.e. Follicule- Stimulating Hormone Receptor (FSHR) and Luteinizing-Hormone and Human Chorionic Gonadotropin Receptor (LHCGR).
In mammals, the expression of both receptors was traditionally considered gonad-specific and cell-specific. However, recent studies demonstrated that gonadotropin receptors are also located in both non-reproductive and reproductive extragonadal tissues.
Among the extragonadal reproductive tissues, the endometrium is one of the most studied due to his critical role during the embryo implantation once that in vitro fertilization (IVF) occurs. At the same time the side effects due to hormonal protocol for IVF on endometrium is poor understood.
At today the FSHR and LHCGR presence and their role in the endometrial physiology and pathologies is still under debate. While the studies about the FSHR presence in human endometrium are plain but few, the LHCGR endometrial expression is more controversial since some studies has shown LHCGR expressed in all endometrial phases (proliferative, early mid- and late- secretory and deicidual) and other failed to show its presence.
Moreover, no data have been provided about endometrial FSHR and LHCGR functional characterization and how they affect the endometrial survival although hypothesis has been formulated.
In these study samples of proliferative endometrium (n=12) were cultured and incubated separately with no hormones (negative control) or with 10 ng/ml dosage of gonadotropin recombinant FSH (rFSH), recombinant LH (rLH) and recombinant hCG (rhCG) alone or in combination.
Presence of FSHR and LHCGR in untreated endometrium was confirmed by both RT-PCR and immunohistochemistry. Evaluation of receptors functionality were performed analyzing the expression of aromatase (CYP19A)1 and cytochrome P450 cholesterol side chain cleavage (P450scc) genes, which expression is notoriously affected by gonadotropins treatments. The presence of both genes was initially demonstrated by RT-PCR in untreated endometrium while their expression was investigated by RT-qPCR. In order to analyze the effects of gonadotropins on the endometrial survival, samples of hormone-stimulated endometrium were paraffin included and corresponding sections were immunostained using primary antibodies anti- microtubule associated protein light chain 3α (MAP-LC3α), considered a marker of autophagy, and anti- mammalian homologs of the Ced-4 protein (Apaf-1) assumed as apoptosis marker. In addition, gonadotropins effects on MAP-LC3α gene expression were quantified by RT-qPCR after normalization by the housekeeping gene ribosomal protein S7 (RpS7).
The results obtained with this thesis confirm evidences previously reported about the presence of FSHR and LHR receptors at both protein and gene level in human endometrium. Moreover, this study shows that gonadotropins, reasonably acting through their specific receptor, are able to modulate the endometrium survival. In the specific, while rFSH alone strongly induced autophagy after 24 hours treatment, the other gonadotropins tested (rLH and rhCG) either alone or in combination with rFSH did not affect the expression of MAP-LC3α autophagy marker.
These results shows that gonadotropins treatment are able to affect the endometrium quality and offer new information for to better address IVF/embryo implantations protocols in the future.
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Abstract
The glycoprotein hormones Follicule-Stimulating Hormone (FSH), Luteinizing Hormone (LH) and human Chorionic Gonadotropin (hCG) are fundamental for the regulation of gonadal function and exert their biological function by directly binding to their specific receptor, i.e. Follicule- Stimulating Hormone Receptor (FSHR) and Luteinizing-Hormone and Human Chorionic Gonadotropin Receptor (LHCGR).
In mammals, the expression of both receptors was traditionally considered gonad-specific and cell-specific. However, recent studies demonstrated that gonadotropin receptors are also located in both non-reproductive and reproductive extragonadal tissues.
Among the extragonadal reproductive tissues, the endometrium is one of the most studied due to his critical role during the embryo implantation once that in vitro fertilization (IVF) occurs. At the same time the side effects due to hormonal protocol for IVF on endometrium is poor understood.
At today the FSHR and LHCGR presence and their role in the endometrial physiology and pathologies is still under debate. While the studies about the FSHR presence in human endometrium are plain but few, the LHCGR endometrial expression is more controversial since some studies has shown LHCGR expressed in all endometrial phases (proliferative, early mid- and late- secretory and deicidual) and other failed to show its presence.
Moreover, no data have been provided about endometrial FSHR and LHCGR functional characterization and how they affect the endometrial survival although hypothesis has been formulated.
In these study samples of proliferative endometrium (n=12) were cultured and incubated separately with no hormones (negative control) or with 10 ng/ml dosage of gonadotropin recombinant FSH (rFSH), recombinant LH (rLH) and recombinant hCG (rhCG) alone or in combination.
Presence of FSHR and LHCGR in untreated endometrium was confirmed by both RT-PCR and immunohistochemistry. Evaluation of receptors functionality were performed analyzing the expression of aromatase (CYP19A)1 and cytochrome P450 cholesterol side chain cleavage (P450scc) genes, which expression is notoriously affected by gonadotropins treatments. The presence of both genes was initially demonstrated by RT-PCR in untreated endometrium while their expression was investigated by RT-qPCR. In order to analyze the effects of gonadotropins on the endometrial survival, samples of hormone-stimulated endometrium were paraffin included and corresponding sections were immunostained using primary antibodies anti- microtubule associated protein light chain 3α (MAP-LC3α), considered a marker of autophagy, and anti- mammalian homologs of the Ced-4 protein (Apaf-1) assumed as apoptosis marker. In addition, gonadotropins effects on MAP-LC3α gene expression were quantified by RT-qPCR after normalization by the housekeeping gene ribosomal protein S7 (RpS7).
The results obtained with this thesis confirm evidences previously reported about the presence of FSHR and LHR receptors at both protein and gene level in human endometrium. Moreover, this study shows that gonadotropins, reasonably acting through their specific receptor, are able to modulate the endometrium survival. In the specific, while rFSH alone strongly induced autophagy after 24 hours treatment, the other gonadotropins tested (rLH and rhCG) either alone or in combination with rFSH did not affect the expression of MAP-LC3α autophagy marker.
These results shows that gonadotropins treatment are able to affect the endometrium quality and offer new information for to better address IVF/embryo implantations protocols in the future.
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