Endometriosis and adenomyosis are two frequent diseases closely linked, characterized by ectopic endometrium. Despite their benign nature, endometriosis and adenomyosis impair women's quality of life ...by causing pain and infertility and an increase in the incidence of gynecological malignancies has been reported. Since the first description of ectopic endometrium in 1860, different attempts have been made to describe, classify and understand the origin of these diseases. Several theories have been proposed to describe the pathogenic mechanism leading to the development of adenomyosis or endometriosis. However, all the hypotheses show some limitations in explaining all the different aspects and manifestations of these diseases. Despite the remarkable progress made over recent years, the pathogeneses of endometriosis and adenomyosis remain unclear. Moreover, because of the lack of standardized protocols and diagnostic criteria in pathology practice it is difficult to study and to classify these disorders. The goal of this review is to summarize the pathological aspects of adenomyosis and endometriosis, spanning a historical perspective to newly reported data.
Do platelets aggregate in adenomyotic lesions and participate in adenomyosis pathogenesis and related fibrosis?
Eutopic and ectopic endometrium from 17 patients with adenomyosis and endometrium from ...23 healthy controls were collected. Immunohistochemical analyses of platelet marker CD41, transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF) were performed to investigate aggregation and activation of platelets in the stroma. Picrosirius staining was carried out to evaluate the extent of fibrotic tissue.
Stroma in the control group showed higher CD41 staining levels than ectopic stroma from patients with adenomyosis (P < 0.001). In patients with adenomyosis, eutopic stroma expressed more extensive CD41 staining than ectopic stroma (P < 0.0001). Stroma in the control group exhibited higher TGF-β1 expression than eutopic and ectopic stroma from adenomyosis patients (P = 0.009 and P < 0.0001). Stroma in the control group also expressed higher VEGF levels than ectopic stroma from patients with adenomyosis (P < 0.001). In patients with adenomyosis, eutopic stroma showed higher VEGF expression than ectopic stroma (P = 0.021). Stroma in ectopic endometrium from adenomyosis patients displayed greater Picrosirius staining compared with both eutopic stroma from adenomyosis patients and stroma in the control group (P < 0.0001).
The results of this study did not detect a primary role for platelet activation or aggregation in the pathophysiological process of adenomyosis. Higher rates of collagen fibres were found in adenomyotic lesions, likely to be related to a TGF-β1-independent pathway. Collagen fibre deposition was more extensive in adenomyotic lesions, consistent with fibrosis.
Purpose
To investigate whether mitochondrial content, activity, and morphology differ in prepubertal versus adult ovarian follicles.
Methods
Ovarian tissue was collected from 7 prepubertal girls (age ...1–10 years) and 6 adult women (age 20–35 years). Primordial and primary follicles were isolated from frozen-thawed prepubertal and adult ovarian tissue and their viability was assessed. Mitochondrial content was investigated by TOMM20 immunostaining of prepubertal and adult ovarian tissue, while mitochondrial activity in isolated follicles was analyzed by MitoTracker CM-H2XRos and JC-1. Frozen-thawed ovarian tissue from the same patients was also evaluated by transmission electron microscopy to examine mitochondrial morphology.
Results
Higher TOMM20 staining was detected in prepubertal follicles compared to their adult counterparts, indicating the presence of more mitochondria in prepubertal follicles. Analysis of mitochondrial activity by MitoTracker showed higher fluorescence intensity in prepubertal follicles, suggesting that follicles in this group are more active than adult follicles. JC-1 analysis did not reveal any statistically significant difference in the inactive/active ratio between the two groups. Moreover, ultrastructural analysis by TEM detected morphological differences in the shape and cristae of prepubertal mitochondria, probably suggesting a mechanism of response to autophagy.
Conclusion
Differences in the number, activity, and morphology of mitochondria were reported, suggesting that consequential modifications might occur during puberty, which could be the window of opportunity required by mitochondria to undergo changes needed to reach maturity, and hence the capacity for ovulation and fertilization.
Vitrification and xenografting of human ovarian tissue Amorim, Christiani Andrade, V.M.D., Ph.D; Dolmans, Marie-Madeleine, M.D., Ph.D; David, Anu, Ph.D ...
Fertility and sterility,
11/2012, Letnik:
98, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Objective To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. Design Pilot study. Setting Gynecology research unit ...in a university hospital. Patient(s) Ovarian biopsies were obtained from seven women aged 30–41 years. Intervention(s) Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. Main Outcome Measure(s) The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. Result(s) After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)–warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. Conclusion(s) Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.
Graves' disease (GD) is an autoimmune thyroiditis often associated with Graves' orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We ...investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.
Research question
Are glioma-associated oncogene homolog 1, 2, and 3 (GLI1, 2, and 3) and protein patched homolog 1 (PTCH1) specific markers for precursor theca cells in human ovaries as in mouse ...ovaries?
Design
To study the GDF9-HH-GLI pathway and assess whether GLI1 and 3 and PTCH1 are specific markers for precursor theca cells in the human ovary, growth differentiation factor 9 (GDF9), Indian Hedgehog (IHH), Desert Hedgehog (DHH), Sonic Hedgehog (SHH), PTCH1 and GLI1, 2 and 3 were investigated in fetal (
n
=9), prepubertal (
n
=9), reproductive-age (
n
=15), and postmenopausal (
n
=8) human ovarian tissue. Immunohistochemistry against GDF9, IHH, DHH, SHH, PTCH1, GLI1, GLI2, and GLI3 was performed on human ovarian tissue sections fixed in 4% formaldehyde and embedded in paraffin. Western blotting was carried out on extracted proteins from the same samples used in the previous step to prove the antibodies’ specificity. The quantitative real-time polymerase chain reaction was performed to identify mRNA levels for
Gdf9
,
Ihh
,
Gli1
,
Gli2
, and
Gli3
in menopausal ovaries.
Results
Our results showed that, in contrast to mice, all studied proteins were expressed in primordial follicles of fetal, prepubertal, and reproductive-age human ovaries and stromal cells of reproductive-age and postmenopausal ovaries. Intriguingly,
Gdf9
,
Ihh
, and
Gli3
mRNA, but not
Gli1
and
2
, was detected in postmenopausal ovaries. Moreover, GLI1, GLI3, and PTCH1 are not limited to a specific population of cells. They were spread throughout the organ, which means they are not specific markers for precursor theca cells in human ovaries.
Conclusion
These results could provide a basis for understanding how this pathway modulates follicle development and ovarian cell steroidogenesis in human ovaries.
Increasing imaging data point to a link between deep endometriotic nodules (DENs) and uterine adenomyosis (AD). The study aimed to investigate this link at the histological level and detect potential ...features shared by the two diseases. We collected formalin-fixed paraffin-embedded tissue (endometrium and lesions) from women with DENs of the rectovaginal septum (
= 13), AD (
= 14), and control subjects (
= 14). Immunohistochemical analyses of CD41 and CD68 were conducted to explore the roles of platelets and macrophages, respectively. Picrosirius red staining was carried out to gather evidence of fibrosis. Vascular endothelial growth factor (VEGF) was assessed, and total numbers of CD31-positive vessels were calculated to investigate the mechanism governing angiogenesis. Double immunohistochemistry for CD31 and alpha smooth muscle actin (αSMA) was performed to discern stable vessels. Platelet aggregation was significantly decreased in both types of lesions compared to their corresponding eutopic endometrium and healthy controls. Macrophage numbers were higher in both lesions than in their corresponding endometrium and healthy subjects. Significantly higher rates of collagen accumulation were detected in DENs and AD lesions compared to their corresponding eutopic and healthy endometrium. VEGF expression was downregulated in the stromal compartment of AD lesions compared to the healthy endometrium. The total number of vessels per area was significantly higher in DENs and AD lesions than in the healthy endometrium. Rates of αSMA-surrounded vessels were decreased in DENs and AD lesions compared to their corresponding eutopic and healthy endometrium. We report common pathogenic mechanisms between DENs and AD, namely excessive macrophage accumulation, fibrosis, and irregular angiogenesis. Our results further support the notion of DENs and AD being linked at the histological level.
Objective To set up a protocol to isolate human preantral follicles with an enzyme produced in good manufacturing practice conditions for use in a clinical setting. Design For follicle isolation, ...ovarian biopsies were divided into two halves: one was treated with collagenase IA and the other with Liberase DH (Dispase High) Research Grade. Setting Academic research unit. Patient(s) Twelve women undergoing laparoscopy for benign gynecological disease. Intervention(s) Follicle isolation. Main Outcome Measure(s) Follicles were counted, their morphology was analyzed, and follicular viability was evaluated by live/dead assays before and after 7 days of in vitro culture. Their structural preservation was assessed after isolation by electron microscopy. Result(s) A total of 1,030 follicles were obtained after isolation: 566 with Liberase DH and 464 with collagenase IA. The percentage of viable follicles (with <10% of dead granulosa cells GC) was not found to be statistically different before or after culture in either group (Liberase DH: 95% and 81%, respectively; collagenase: 96% and 87%, respectively). A significant increase in follicle size was observed in both groups after culture. Liberase DH did not affect the ultrastructure of isolated follicles. Conclusion(s) Liberase DH allows isolation of a high number of preantral follicles, maintaining their viability, even after in vitro culture, and their ultrastructure. In addition, Liberase DH can be produced in good manufacturing practice conditions, allowing use of isolated preantral follicles for future clinical applications.
Objective To analyze the ultrastructure of human ovarian follicles after cryopreservation and short-term xenografting. Design Prospective experimental study. Setting Academic gynecology and anatomy ...research units. Patient(s) Ovarian cortical biopsy specimens were obtained from 13 patients. Intervention(s) Each ovarian biopsy specimen was dissected into pieces of 1 mm3 and divided into three groups: 1 fresh tissue, 2 frozen-thawed tissue, and 3 frozen-thawed tissue xenografted onto the peritoneum of nude mice for 3 weeks. Main Outcome Measure(s) Follicular ultrastructure was assessed by light and transmission electron microscopy in 1 fresh, 2 frozen, and 3 frozen-grafted tissue. Result(s) Thirty-five ovarian follicles were analyzed by light and transmission electron microscopy. Twenty-five primordial and primary ovarian follicles were found. Most of them exhibited ultrastructurally well preserved features (fresh N = 8/10, frozen N = 7/10, and frozen-grafted N = 4/5 tissue). Ten secondary follicles were present in xenografts. By transmission electron microscopy, all the healthy-looking secondary follicles (70%) were shown to contain intact oocytes, with features typical of earlier developmental stages, surrounded by several layers of follicular cells. Conclusion(s) The present study demonstrates, for the first time, that cryopreservation and xenotransplantation do not appear to greatly affect human primordial/primary follicle ultrastructure. Interestingly, in frozen-thawed xenografts, secondary human ovarian follicles presented a well preserved ultrastructure, but asynchrony between oocyte and granulosa cell development was detected. The possible causes for this asynchrony are discussed.
This study was designed to evaluate follicular survival and growth after short-term transplantation of fresh isolated human follicles and ovarian cortical tissue to nude mice. Ovarian biopsies were ...obtained from nine women undergoing laparoscopy. Twelve nude mice were xenografted with an ovarian cortical fragment in the right ovarian bursa, and a clot containing isolated follicles in the left, for a period of 7 days. One ungrafted fragment was used as a control. Histological sections were analyzed to determine follicle number and stage. The proliferative status of follicular cells was assessed by Ki-67 immunostaining. A total of 659 follicles was analyzed by histology and 545 follicles by immunohistochemistry. The percentage of primordial follicles was found to be markedly reduced 1 week post-grafting when compared with ungrafted tissue, while the percentage of primary follicles had significantly increased. Only 8% of follicles showed Ki-67-positive granulosa cells before grafting, whereas 1 week after grafting, 71% of follicles in fragments and 67% of isolated follicles were Ki-67-positive (P<0.001). Moreover, the histological aspect of isolated follicle grafts was similar to that of grafted fragments: follicles were surrounded by vimentin-positive stroma-like tissue of human origin, as confirmed by fluorescent in situ hybridization with human-specific probes. Our results demonstrate, for the first time, that isolated human follicles are able to survive and grow after xenografting. This study also shows massive in vivo follicular activation after transplantation of grafted fragments and isolated follicles. One week after grafting, well-structured stroma-like tissue of human origin was observed around the isolated follicles. The potential origin of this stroma is discussed.
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