Frank Manconi

3-Dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.

Dendritic cell populations in the eutopic and ectopic endometrium of women with endometriosis

BACKGROUND Immune alterations may be involved in the pathogenesis and progression of endometriosis. Dendritic cells (DCs) are potent antigen presenting cells that are highly involved in the initiation of the immune response. The aim of this study was to investigate DC populations in the eutopic and ectopic endometrium of women with endometriosis compared with controls. METHODS Hysterectomy samples were obtained from premenopausal women with (n = 33) and without (n = 28) endometriosis. In addition, paired peritoneal endometriotic lesions and uterine curettings were collected from 32 women with endometriosis. Specimen sections were stained immunohistochemically using antibodies for monoclonal mouse antibodies directed against human CD1a and CD83, which are specific for immature and mature DCs, respectively. RESULTS The mean density of endometrial CD1a+ DCs in the basal layer was significantly increased in women with endometriosis compared with controls during the proliferative phase only (P = 0.001). There was a highly significant decrease in the density of endometrial CD83+ DCs in women with endometriosis compared with controls in both layers of the endometrium across all phases of the menstrual cycle (P = 0.001). The density of CD1a+ DCs was significantly increased in peritoneal endometriotic lesions (P = 0.003) and in the surrounding peritoneum (P = 0.001) compared with paired uterine curettings and peritoneum distant from the lesion. CONCLUSIONS Both CD1a+ and CD83+ DC populations were altered in the eutopic and ectopic endometrium of women with endometriosis compared with controls. Alterations in these cells, which play a crucial role in the coordination of the immune response, may be involved in pain generation and the pathogenesis of endometriosis.

Endometrial dendritic cell populations during the normal menstrual cycle

BACKGROUND Dendritic cells (DCs) are specialized antigen presenting cells that are highly involved in the stimulation and modulation of the immune response within mucosal surfaces, including the female reproductive tract. DCs have been poorly characterized in the non-pregnant endometrium. METHODS Hysterectomy specimens were obtained from premenopausal women (n = 49) with histologically normal endometrium. Endometrial sections were stained immunohistochemically using antibodies for monoclonal mouse anti-human CD1a and CD83, two markers which are specific for populations of immature and mature DCs, respectively. RESULTS There was a significantly higher density of endometrial CD1a+ DCs than CD83+ DCs throughout the menstrual cycle (P < 0.001). The density of CD1a+ and CD83+ DCs did not vary between the fundus and isthmus of the uterus. There was a significant increase in the density of CD1a+ DCs, but not CD83+ DCs, in the basal layer of the endometrium through the phases of the menstrual cycle. The density of CD83+ was significantly greater in the basal layer compared with the functional layer during both the proliferative (P = 0.004) and secretory phases (P = 0.001), whereas for CD1a+ DCs, the greater density in the basal layer was only observed in the secretory phase (P < 0.001). CONCLUSIONS The highly coordinated cyclical changes in DC populations during the normal menstrual cycle reported in this study may be important for local regulatory mechanisms relevant to menstruation and implantation; alterations in this normal profile may contribute to the development of disturbances of function, fertility and even benign gynaecological disease.

Uterine and Placental Angiogenesis in the Australian Skinks, Ctenotus taeniolatus, and Saiphos equalis

The evolution of viviparity requires modifications to multiple integrated physiological features to support embryonic development during pregnancy. Embryonic growth during pregnancy is dependent upon the capacity of the uterine vascular system to satisfy increasing embryonic oxygen demand throughout gestation. We tested the hypothesis that total surface area of uterine blood vessels increases in concert with embryonic growth, and hence its oxygen demand, during gestation. We used immunofluorescence and laser‐scanning confocal microscopy to quantify uterine microvascular density and morphology during gestation in the oviparous skink Ctenotus taeniolatus and in Saiphos equalis, a skink species with prolonged egg retention. For C. taeniolatus, vessel density (Nv) and vessel length‐density (Lv) in the embryonic hemisphere of the uterus is 23% and 17% less, respectively, than that of S. equalis and vascular surface‐area does not differ as a function of embryo stage. For S. equalis, overall Nv, Lv, and vessel diameter (Dv), does not change during the first half of gestation but increases by 36% (Nv), 44% (Lv), and 60% (Dv) by near‐term embryo stages late in gestation. The chorioallantoic membrane of S. equalis increases in absolute size but vascular density does not differ as a function of embryo stage. The marked increase in uterine vascular density during late gestation coincides with the phase of rapid growth in embryo mass and concomitant increase in metabolic rate. Expansion of the uterine vascular bed in concert with embryo size and metabolism is likely to be an important transitional step in the evolution of viviparity. Anat Rec, 293:829–838, 2010.

Quantitative measurements of menstrual blood loss in ovulatory and anovulatory cycles in middle- and late-reproductive age and the menopausal transition.

OBJECTIVE: To measure menstrual blood loss before and during the menopausal transition and to explore the relationships between menstrual blood loss and menstrual cycle irregularity and reproductive hormone levels. METHODS: Two consecutive menstrual blood loss measurements were performed in 77 healthy women aged 21-55 years, classified as midreproductive age (n=21, control group), late-reproductive age (n=17), early-menopausal transition (n=16), and late-menopausal transition (n=23). Serum hormone levels (estradiol [E2], progesterone, follicle-stimulating hormone, luteinizing hormone, and inhibins) were measured three times per week from the start of one menstrual period to the end of the subsequent menstrual period. RESULTS: There were nine, one, zero, and two anovulatory cycles captured in the late-menopausal transition, early-menopausal transition, late-reproductive age, and midreproductive age groups, respectively. The median (range) menstrual blood loss values after ovulatory cycles were 30 (142), 33 (147), 55.7 (105), and 68.9 (234) mL in the midreproductive age, late-reproductive age, early-menopausal transition, and late-menopausal transition groups, respectively. After anovulatory cycles in the late-menopausal transition group, menstrual blood loss was only 11.8 (97) mL. In the late-menopausal transition group, menstrual blood loss after an ovulatory cycle was significantly higher than when occurring after an anovulatory cycle (P=.008, Kruskal-Wallis). The highest menstrual blood loss measurements were in women in the late-menopausal transition group who experienced ovulatory cycles with abnormally high E2 levels and disturbed E2 secretion patterns. CONCLUSION: The onset of variability in menstrual blood loss was associated with the onset of irregular cycles. Excessive menstrual blood loss (greater than 250 mL) was associated with ovulatory cycles with high E2 levels and late menopausal transition. LEVEL OF EVIDENCE: III

Computer-generated, three-dimensional reconstruction of histological parallel serial sections displaying microvascular and glandular structures in human endometrium

This paper describes a technique to develop high-resolution three-dimensional (3D) images of microvasculature structures in curettage, hysterectomy or endometrial resection biopsies using parallel histological serial sections. Employing a labelled streptavidin-biotin-alkaline phosphatase (LSAB(+)) method and visualising by using DAB(+) with the primary antibody, mouse anti human Q-Bend-10, the images were directly digitised from a light microscope into the KS400 Universal Image Processing and Analysis software via a CCD colour camera; binary images of the structures were created and the binary images were exported into VoxBlast 3D rendering software to view still and rotating 3D images on a computer monitor. This in turn enabled hard copies of the full sequence to be printed.

Second harmonic imaging of collagen in mammalian tissue

It has recently been demonstrated that collagen is a very effective upconverter of light by second harmonic generation (SHG) but hitherto the potential this offers for biomedical imaging has not been realized. We show that bright SHG images van be obtained over a wide excitation range at illumination levels comparable to or lower than those required for two-photon excitation of fluorescent labels, with no damage to the collagen structure. Both paraffin and cryostat sections have been used, and medically significant results have been obtained in several fields. We show that the signal is easily distinguished from single and two-photon excited fluorescence by its forward propagation and narrow spectral width; in principle it could also be distinguished by lifetime. Key microscope requisites are: immersion objectives and condensers, high-efficiency PMT detectors for transmitted light, suitable filters, and effective blocking of stray light, especially from the mercury lamp.

Novel finding of high density of activated mast cells in endometrial polyps.

Endometrial polyps are benign lesions frequently identified in women with infertility or abnormal uterine bleeding in the reproductive and postmenopausal phases We report the striking observation that the numbers of activated mast cells expressing tryptase are increased more than sevenfold throughout the cycle in endometrial polyps (n = 20) compared with normal endometrium. This novel finding has important implications for growth, development, and symptoms associated with polyps in many different tissues.

Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis

To study the density of nerve fibers in cases of deep infiltrating endometriosis (DIE) of the rectovaginal septum in relation to various clinical factors.

Microanatomy and function of the eutopic endometrium in women with endometriosis

Endometriosis is a disease that still presents many puzzles to clinicians and basic research scientists. Until recently, it has been regarded as a condition that arises when normal endometrium adheres to the peritoneal surface and then grows into an ‘inflammatory’ lesion, which adversely influences local reproductive tract function and causes pain. It is now becoming clear that the endometrium within the uterus in these women differs significantly in function from ‘normal’, and that these anomalies probably precede the development of classical ectopic endometriotic lesions. However, the etiology and mechanisms by which endometriosis arises are still far from certain. This review attempts to address the extensive, but fragmented, evidence that demonstrates widespread molecular disturbances in endometrial function and microstructure underlying this complex condition. The review also addresses some of the novel concepts that are being raised by these exciting new discoveries.