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

OBJECTIVE: To study the density of nerve fibers in cases of deep infiltrating endometriosis (DIE) of the rectovaginal septum in relation to various clinical factors. DESIGN: A research laboratory-based study. SETTING: A tertiary center together with a research laboratory. METHODS: Archived DIE tissue samples from 45 women operated upon for rectovaginal septum DIE were re-examined histologically, and by immunohistochemistry. MAIN OUTCOME MEASURES: The effect of progestogens or combined oral contraceptives on the density of nerve fibers, and the expression of nerve growth factor (NGF) and its high-affinity receptor (tyrosine kinase receptor A, Trk-A). RESULTS: The use of hormonal therapy was associated with reduced densities of sympathetic, parasympathetic and sensory nerve fibers in DIE lesions. Density of total nerve fibers (with pan-neuronal marker PGP9.5) was significantly lower (p < 0.05) in lesions collected from hormone-treated women (8.6/mm², 4.2-20.8/mm²; median density, from 25th to 75th quartiles) compared with that in lesions from untreated women (24.9/mm², 11.2-34.9/mm²). DIE lesions stained strongly for NGF and its receptor Trk-A. Expression of NGF, but not of Trk-A, was significantly reduced during use of hormonal therapy. CONCLUSIONS: Use of hormonal therapy was associated with significantly reduced nerve fiber density in DIE lesions. This may be an important mechanism of action of hormonal therapy for controlling DIE pain symptoms. The expression of estrogen-regulated NGF and its receptor was only partially suppressed during the use of hormonal therapy, suggesting that local estrogen action is often maintained during conventional hormonal therapy in cases of DIE.

A historical overview of the study and representation of uterine microvascular structures.

The concept of anatomical modelling of the internal vascular structures of organs dates back to the Middle Ages by way of corrosion casting. The first to apply this classic injection technique in the reproductive arena was John Hunter (1754), who undertook to establish the independence of the maternal and fetal circulations in the placenta. The first detailed microscopic study of the endometrial vessels was undertaken a century later. Endometrial inoculation studies in the 1930s with coloured fluids such as India ink have provided the basis of our current understanding of the complex sequence of morphological vascular changes which occur in the endometrial tissue leading up to and during the process of menstruation. Classic injection techniques were limited in that they were often associated with artefacts due to injection-induced vessel breakages and variability in size of the suspended particles in the injection material. Following this, the smallest blood vessels were better demonstrated using Gomori's alkaline phosphate method. An adaptation of this method in the early 1960s demonstrated the uterine vasculature in a more detailed way than ever before. In the early 1970s, novel microradiography studies involved the injection of warmed radio-opaque medium into both arterial and venous microvasculature of the human uterus. Early 1980s investigators also utilized corrosion casting of uterine microvessels combined with scanning electron microscopy. The last 20 years have seen the dawn of the computer age, immunohistochemistry, advanced microscopy (laser scanning confocal and multiphoton emission), and stereological methods to obtain quantitative measurements of 3-dimensional endometrial vascular structures. This review article contains a historical overview of uterine microanatomical vascular visualisation from the early beginnings to the latest computerised techniques.

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.

The Expression and Cellular Localisation of Neurotrophin and Neural Guidance Molecules in Peritoneal Ectopic Lesions.

Endometriosis is a gynaecological disorder characterised by the presence of endometrial-like tissue outside the uterus. It affects 10-15% of women during their reproductive age. The existence of close and complex relationship between chronic pelvic pain and endometriosis are widely recognised. However, the mechanisms of pain generation in women with endometriosis remain poorly understood. Immunohistochemistry was used to assess the density of nerve fibres stained with protein gene product 9.5 (PGP9.5) and the expression of various neurotrophins including glial cell derived neurotrophic factor (GDNF), persephin, neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and neuronal guidance molecules semaphorin 3E and Slit-2 and their receptors Plexin-D1 and Robo4 in peritoneal ectopic lesions from women with endometriosis and uninvolved peritoneum samples. Neurotrophins and neuronal guidance molecules and their receptors are synthesised in situ within peritoneal ectopic lesion which suggest their role in facilitating and maintaining the growth of nerve fibres. These molecules were found to be overall most highly expressed in the glands of endometriotic peritoneal lesions. In addition, the presence of ectopic lesions within the peritoneal cavity may affect the environment; in turn, the peritoneum altered appeared to play a role in the growth of nerve fibres and their development and maintenance in peritoneal lesions. Through exploring different neuronally active factors in and around ectopic lesions which may be contributing to pain generation, this study provides an insight and better understanding of the pain mechanisms associated with peritoneal endometriosis.

Endometrial polyps: Pathogenesis, sequelae and treatment.

Endometrial polyps are overgrowths of endometrial glands that typically protrude into the uterine cavity. Endometrial polyps are benign in nature and affect both reproductive age and postmenopausal women. Although endometrial polyps are relatively common and may be accompanied by abnormally heavy bleeding at menstruation. In asymptomatic women, endometrial polyps may regress spontaneously, in symptomatic women endometrial polyps can be treated safely and efficiently with hysteroscopic excision.

Endometrial thickness measured by ultrasound scan in women with uterine outlet obstruction due to intrauterine or upper cervical adhesions.

BACKGROUND: A subgroup of women with Asherman's syndrome has adhesions of limited extent completely blocking the lower uterine cavity or upper cervix, whereas the upper endometrium remains normal. Haematometra are rarely found in these women. We tested the hypothesis that women with localized adhesions occluding the uterine outlet (but not affecting the upper uterine cavity) will have much thinner endometrium than controls. METHODS: Twenty-six women with Asherman's syndrome (16 with limited outlet adhesions only) and 50 with normal menstrual cycles underwent transvaginal ultrasound scan where endometrial double thickness was measured precisely and the cycle phase assessed. The presence of any fluid in the uterine cavity was noted. RESULTS: The endometrium in women with Asherman's syndrome, in whom uterine outlet blockage was the sole abnormality (subgroup 3), was substantially thinner (mean +/- SEM: 3.9 +/- 0.4 mm) than controls (8.5 +/- 0.05; P < 0.001), and haematometra were very uncommon (1 of 16). Endometrial thickness at all stages of the ovarian/menstrual cycle in all three subgroups of Asherman's syndrome was significantly less than in normal menstruating controls. CONCLUSIONS: Non-invasive ultrasound measurements have demonstrated very thin endometrium and absence of haematometra in most women with uterine outlet occlusion by adhesions. This unusual phenomenon of failure of cyclical endometrial growth and breakdown in the sole presence of cervical occlusion by adhesions merits further study.

Blood microvasculature and lymphatic densities in endometrial polyps and adjacent and distant endometrium.

OBJECTIVE: Endometrial polyps are localised growths of endometrial tissue containing glands, stroma and blood vessels, covered with epithelium. The reported prevalence of endometrial polyps is dependent upon the population being studied and the uterine imaging technique utilised. The light microscopy literature provides very little information regarding their microvasculature and lymphatic systems; however, a plethora of ultrasound data demonstrating single central arteries in most medium- or large-sized endometrial polyps are well documented. METHODS: Archived formalin-fixed paraffin-embedded blocks of endometrial curettings were retrieved from files for women with confirmed endometrial polyps (n = 20) and women with normal endometrium (control endometrium; n = 32). Immunohistochemistry was performed with the antibodies CD31 (blood vessels) and D2-40 (lymphatics). Blood vessels and lymphatics were quantified in endometrial polyps and adjacent, distant and control endometrium. RESULTS: CD31 and D2-40 staining was present in all specimens, although there were no significant differences in blood vessel (F(3,70) = 2.36, p = 0.079) and lymphatic (F(3,70) = 0.16, p = 0.920) densities between endometrial polyps as well as adjacent, distant and control endometrium. There were also no significant differences in women with endometrial polyp-associated bleeding and those with no bleeding. In relation to infertility, there were no significant differences found in blood and lymphatic densities between women with endometrial polyps who were infertile and those with endometrial polyps who were fertile. CONCLUSION: Small blood vessel wall and perivascular structures rather than the distribution of vessels may be associated with abnormal bleeding.

Three dimensional microvascular measurements in human endometrium using optical slices from laser scanning confocal microscopy (LSCM).

There is increasing interest in the structure of the microvascular environment in human endometrium because of the recognition of the complexity and functional importance of this tissue. Endometrial microcirculatory networks and their relationships have rarely been studied in three-dimensions. Longitudinal uterine slices containing endometrial tissue were carefully selected from women undergoing a hysterectomy. Formalin-fixed endometrial sections (≤ 50 µm) representing the fundal and isthmic regions were immunofluorescently labeled with monoclonal antibody (CD34) to target the endothelium of microvessel and fluorescein isothiocyanate (FITC) labeled goat anti-mouse. Digital images were acquired using a Nikon Eclipse E800 microscope equipped with a Radiance 2000 confocal scanning laser attachment. ImarisBasic 4.1 visualization suite was utilized for qualitative interpretation. NeuronTracer 1.0 software was utilized to derive the length and numerical densities. There were significant changes across the phases of the menstrual cycle in functional and basal endometrial layers in vessel length density (LD(v)) and branch point density (ND(v)) within both fundal and isthmic regions of the uterus (P<0.001). There was also a significant effect of menstrual cycle phase on mean vessel segment length (SL(v)) within each region and within each of the layers (P<0.001). The capillary radial diffusion distance r(diff) was negatively correlated with LD(v). In general, within each of the menstrual cycle phases, LD(v), ND(v) were greater in the fundal than the isthmic regions while, in contrast, SL(v) was found to be greatest in the isthmic region. Utilization of immunofluorescence and laser scanning confocal microscopy has enabled us to demonstrate significant vascular changes in human endometrial layers illustrating that in general, within each of the menstrual cycle phases, vessel length and branch point densities were greater in the fundal than the isthmic regions, while vessel segment lengths were found to be greatest in the isthmic region.

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.

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.

Timing and indication for curettage after medical abortion in early pregnant women with prior uterine incision.

BACKGROUND: Termination of pregnancy is an important and necessary back-up method for family planning services in many countries. The combination of mifepristone and misoprostol is a widely used alternative to surgical evacuation of the uterus in early pregnancy; however, there are few reports about medical abortion in women with a prior uterine incision and few studies have described curettage occurring as part of the procedure and an indication for the intervention. Curettage in a prior uterine incision can increase operative complications. The purpose of this study was to investigate whether vaginal bleeding intervals, routine ultrasound scan and serum beta-hCG test after medical abortion could accurately identify women with uterine scars who would require curettage. METHODS: Six hundred sixty-eight women with a uterine scar and at up to 49 days of gestation underwent a medical abortion with mifepristone and misoprostol. Each woman took 50 mg and 25 mg of mifepristone orally in the morning and in the evening, respectively, for 2 days and 600 mcg of misoprostol orally on the third day. RESULTS: Of the 668 women, 6 (0.9%) were lost to follow-up. The overall complete abortion rate was 91.7%; 55 women underwent curettage, including 2 women with heavy bleeding, 3 women with ongoing pregnancy and 34 women with incomplete abortion. The incomplete abortion rate was significantly greater in women with persistent bleeding lasting 21 days than in women with persistent bleeding lasting 14 days (p<.001), and the overall sensitivity and specificity of vaginal bleeding interval (21 days) were 97.1% and 75%, respectively. The incomplete abortion rate was also greater in women whose serum beta-hCG was >or=500 IU/L than in women whose serum beta-hCG was <500 IU/L (p<.001), and the overall sensitivity and specificity of serum beta-hCG (>or=500 IU/L) were 97.1% and 62.5%, respectively. Moreover, the incomplete abortion rate was greater in women with an endometrial thickness >or=15 mm than in women with an endometrial thickness <15 mm (p<.001), and the overall sensitivity and specificity of endometrial thickness (>or=15 mm) were 94.1% and 75%, respectively. No complication occurred. CONCLUSIONS: The combination of mifepristone and misoprostol was found to be a safe and effective method to terminate early pregnancy in women with a previous cesarean delivery. If a woman with a prior uterine incision experienced vaginal bleeding intervals >or=21 days and/or had a bilayer endometrial thickness >or=15 mm and/or serum beta-hCG >or=500 IU/L after a medical abortion, then she should undergo curettage.

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.

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.