Clinically Responsive Genomic Analysis Pipelines: Elements to Improve Detection Rate and Efficiency.

Massively parallel sequencing has markedly improved mendelian diagnostic rates. This study assessed the effects of custom alterations to a diagnostic genomic bioinformatic pipeline in response to clinical need and derived practice recommendations relative to diagnostic rates and efficiency. The Genomic Annotation and Interpretation Application (GAIA) bioinformatics pipeline was designed to detect panel, exome, and genome sample integrity and prioritize gene variants in mendelian disorders. Reanalysis of selected negative cases was performed after improvements to the pipeline. GAIA improvements and their effect on sensitivity are described, including addition of a PubMed search for gene-disease associations not in the Online Mendelian Inheritance of Man database, inclusion of a process for calling low-quality variants (known as QPatch), and gene symbol nomenclature consistency checking. The new pipeline increased the diagnostic rate and reduced staff costs, resulting in a saving of US$844.34 per additional diagnosis. Recommendations for genomic analysis pipeline requirements are summarized. Clinically responsive bioinformatics pipeline improvements increase diagnostic sensitivity and increase cost-effectiveness.

Contraception for women with medical disorders.

Many women in the reproductive years have chronic medical conditions that are affected by pregnancy or in which the fetus is placed at increased risk. In most of these women, ongoing medical management of their conditions is greatly improved, even compared with a decade or two ago. However, their condition may still be seriously exacerbated by the physiological changes of pregnancy, and close monitoring of a carefully planned pregnancy is optimal. This requires effective and safe contraceptive use until pregnancy is desired and the medical condition is stabilised. Many contraceptives will also have adverse effects on some medical conditions, and there is now a considerable awareness of the complexities of some of these interactions. For this reason the World Health Organization has developed an excellent, simple and pragmatic programme of guidelines on a four point scale (the WHO "Medical Eligibility Criteria": WHO-MEC), summarising risk of specific contraceptive methods in women with specified chronic medical conditions. The general approach to contraceptive management of many of these conditions is addressed in this article.

Asynchronous glands in the endometrium of women with recurrent reproductive failure.

Of 969 non-consecutive endometrial biopsies performed for investigation of recurrent reproductive failure, 20 cases (2.1%) showed the striking presence of retarded or asynchronous endometrial glands in otherwise unremarkable mid or late secretory endometrium. These glands were characterised by tall columnar cells with crowded nuclei showing increased reactivity for the proliferative marker MIB-1, occasional mitoses, greatly reduced or absent secretion, and persistent expression of oestrogen receptors and usually progesterone receptors and their isoforms typical of late proliferative phase endometrium. The nearby endometrial stromal cells exhibited no discernibly reduced reactivity for calretinin. These changes were seen in single glands (even portions of glands), or clusters of glands, adjacent to normal late secretory type endometrial glands and set in pseudodecidualised stroma characteristic of late luteal phase. Some examples also displayed adjacent glands with intermediate features and it is speculated that firstly, this is a relatively common phenomenon in women with recurrent miscarriage or implantation failure and with an unknown potential to affect implantation. Secondly, it is an intrinsic defect of the endometrium and can occur in sequential endometrial biopsies in the same patient. Thirdly, it differs from previously described patterns of so-called luteal phase defect or deficient secretory phase in that it occurs in the demonstrated presence of adequate progesterone effect on the endometrium and is associated with persistence rather than exaggerated down-regulation of receptors. Nevertheless, supplementary progesterone therapy (vaginal pessaries) for the first trimester appeared to have a beneficial therapeutic effect on reproductive outcome in these patients.

Peripheral and endometrial dendritic cell populations during the normal cycle and in the presence of endometriosis.

BACKGROUND: Dysfunctional immune response may be implicated in endometriosis pathogenesis, and dendritic cells (DC) may play greater roles in this response than previously recognized. This study set out to evaluate peripheral blood and endometrial DC population changes in the presence and absence of endometriosis pathology. METHODS: Endometrial (n = 83) and peripheral blood samples (n = 30) were subjected to immunohistochemical techniques and flow cytometry, respectively, to assess DC populations in women with and without endometriosis. Three circulating DC subsets (MDC1, MDC2 and PDC, expressing CD1c, CD303 and CD141), and late-stage mature endometrial DCs (using DC-LAMP antibody) were investigated. RESULTS: A highly significant reduction in CD1c intensity on MDC1 populations in peripheral blood was observed between normal cycle proliferative and menstrual phases (p = 0.025), but not in women with endometriosis, in whom CD1c intensity was markedly increased at the time of menstruation (p = 0.05). A significant reduction in peripheral blood MDC2 (p = 0.016) and apparent reduction in endometrial DC-LAMP+ DC (trend, p = 0.062) were observed in women with endometriosis compared with controls, consistent with our preliminary DC data. CONCLUSIONS: Cyclical variation in endometrial and circulating DC populations appears to be crucial during normal menstrual cycles and in the establishment of pregnancy. In endometriosis, circulating and endometrial DC populations are significantly dysregulated at a number of levels, and are likely to contribute to inefficient immunological targeting of endometrial fragments shed at menstruation, facilitating their survival and establishment of endometriosis.

Cellular immune environment in endometrial polyps.

STUDY OBJECTIVE: To investigate the immune environment of endometrial polyps (EPs). DESIGN: Prospective case-control study. SETTING: Teaching hospital and university research laboratory. PATIENT(S): Reproductive-age women undergoing hysteroscopy dilation and curettage for benign indications. Samples were collected from women with (n = 23) and without (n = 40) EPs. INTERVENTION(S): Endometrial samples were immunohistochemically stained with antibodies against mast cells (MCs) and regulatory T cells (Tregs). MAIN OUTCOME MEASURE(S): Tryptase+, chymase+, and c-Kit+ MCs and Foxp3+ Tregs were quantified in EPs and polyp-adjacent, polyp-distant, and control endometrium. RESULT(S): Densities of all MC types were highly significantly increased in EPs compared with adjacent, distant, and control endometrium. Chymase+ and c-Kit+ MCs were increased in density in adjacent compared with control endometrium. c-Kit+ MCs were also increased in distant compared with control endometrium. Foxp3+ Treg density was increased in EPs compared with distant and control endometrium and decreased in distant compared with control endometrium. CONCLUSION(S): This study provides novel insights into localized disturbances in the cellular immune environment within EPs consistent with EPs being inflammatory lesions associated with MC overactivity. Tregs are likely to be recruited to EPs in an attempt to suppress the inflammatory process due to the greatly increased presence of MCs. These immunologic disturbances are likely to be involved in the causation of abnormal bleeding and infertility in premenopausal women with EPs, and their role in the pathophysiology requires further research.

Immunology of normal and abnormal menstruation.

Normal menstruation is an inflammatory process, where the endometrial concentrations and functions of several leukocyte types can change greatly through the menstrual cycle, especially during the premenstrual and menstrual phases. These leukocytes probably have a range of functions related to mucosal protection, decidualization, embryo implantation, and the process of menstrual tissue breakdown, repair and remodeling. Some of these leukocyte changes are apparently linked to changes in the pattern of circulating leukocytes. Many immune cells have been identified in the endometrium, and those with most relevance to the processes of menstruation include uterine natural killer cells, macrophages, mast cells, neutrophils, dendritic cells and Tregs. A range of disturbances in endometrial immune cell numbers, distributions and functions, and in a range of different inflammatory and other mediators, have been identified in women with heavy menstrual bleeding or endometriosis. Sufficient evidence exists to implicate these immune changes in some of the functional disturbances and symptoms identified in these women. This field is greatly under-researched, and ripe for the wider application of modern molecular and cellular techniques in human and animal model studies.

Angiogenesis, lymphangiogenesis and neurogenesis in endometriosis.

Endometriosis is a common, benign gynecological disease affecting 10 - 15% of reproductively aged women. It is characterized by the presence of endometrial-like tissue at sites outside the uterus. The most widely accepted theory of endometriosis pathogenesis proposes that shed menstrual endometrium can reach the peritoneum, implant and grow as endometriotic lesions. Angiogenesis, lymphangiogenesis and neurogenesis are implicated in successful ectopic establishment and the generation of endometriosis-associated symptoms. This review considers these processes as they occur in the eutopic endometrium and ectopic endometriotic lesions of women with endometriosis. Their regulation is inter-connected and complex. Dysregulation in endometriosis occurs on a background of accumulating evidence that endometriosis is an endometrial disease with underlying genetic influences and cross talk with endometriotic lesions. Understanding the roles of angiogenesis, lymphangiogenesis and neurogenesis in endometriosis pathophysiology is essential for the development of novel therapeutic approaches.

A novel pilot study of endometrial stromal cells and immune cell populations in sentinel uterine-draining lymph nodes during the menstrual cycle and in endometriosis.

Recent studies suggest that changes in certain uterine immune cell populations in endometrium of women with endometriosis are likely to precede changes at ectopic sites. This preliminary study is a first look into the function of uterine-draining lymph nodes (LNs) during the menstrual cycle and in the presence of endometriosis. Paraffin-embedded obturator LNs were obtained from women with (n = 7, mean age 44.3) and without (n = 9, mean age 38.4) endometriosis, who had undergone hysterectomy for cervical or ovarian cancer and in whom LN involvement was not detected. Immunohistochemical staining for endometrial stromal cells and a range of immune cell populations was performed. The CD10+ endometrial stromal cells were detected in uterine-draining LNs throughout the menstrual cycle with numbers peaking during menstruation. The inflammatory process of menstruation was also associated with increased numbers of CD3+, CD4+, Foxp3+, DC-Sign+, CD68+, CD20+, CD79+, and plasma cells. In endometriosis, CD10+ endometrial stromal cells were further increased in numbers, but CD3+, CD4+, DC-Lamp+, FoxP3+, and plasma cells were reduced. This study indicates that efficient immunological responses may be required to contain shed endometrial fragments within the draining uterine LNs thus preventing their further dissemination with establishment of ectopic lesions at distant sites.

The role of Foxp3+ regulatory T-cells in endometriosis: a potential controlling mechanism for a complex, chronic immunological condition.

BACKGROUND: Endometriosis is an inflammatory condition, associated with highly dysregulated immune response at both uterine and peritoneal levels. Surprisingly, Foxp3+ regulatory T-cells, which control and suppress a range of immune responses, have not previously been investigated in endometriosis. METHODS AND RESULTS: Immunohistochemical analysis of Foxp3+ cells in 127 eutopic endometrial samples and 59 ectopic peritoneal lesions revealed that these immune cell populations are highly disturbed in women suffering from endometriosis. We showed that Foxp3+ cells remained highly up-regulated during the secretory phase of the menstrual cycle, while at this time their expression is significantly down-regulated in women without endometriosis (P < 0.001). Foxp3+ cells were detected in the stroma of 18 of the 59 peritoneal endometriotic lesions, but not in the surrounding or control peritoneal tissue. CONCLUSIONS: We propose that in eutopic endometrium in women with endometriosis Foxp3+ cells decrease the ability of newly recruited immune cell populations to effectively recognize and target endometrial antigens shed during menstruation, allowing their survival and ability to implant in ectopic sites. At these ectopic sites, variable expression of Foxp3+ cells within some peritoneal endometriotic lesions is likely to be linked to the characteristics and stage of individual lesion development and be playing key roles in pathogenesis and progression of this unique condition.

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.

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.

Macrophages and nerve fibres in peritoneal endometriosis.

BACKGROUND: Endometriosis is considered to be an inflammatory disease, and macrophages are the most numerous immune cells in endometriotic lesions. However, the mechanisms underlying the elevation of macrophages and their role in the pathogenesis and manifestations of endometriosis still remain unclear. METHODS: The number of macrophages stained for CD68 in endometriotic lesions (n = 24) and in peritoneum distant from the lesions (n = 14) from women with endometriosis was compared with the number of macrophages in normal peritoneum from women without endometriosis (n = 18). Peritoneal lesions were also double-stained for CD68 and protein gene product 9.5 to study the relationship between macrophages and nerve fibres. RESULTS: The densities of macrophages in peritoneal endometriotic lesions and unaffected peritoneum from women with endometriosis were both significantly higher than that in normal peritoneum from women without endometriosis (P < 0.001). More nerve fibres were also found in the areas where increased numbers of macrophages were identified. CONCLUSIONS: There was a significant elevation of macrophages in both normal peritoneum and peritoneal lesions from women with endometriosis compared with normal peritoneum from women without endometriosis. These cells may well play roles in the growth and development of endometriotic lesions and in the generation of pain through interaction with nerve fibres.

Macrophage expression in endometrium of women with and without endometriosis.

BACKGROUND: Endometriosis is an inflammatory condition, characterized by the presence of endometrial-like tissue outside the uterus. The immune system provides a defence mechanism in response to foreign pathogens, and macrophages play important roles in this response. Activation of macrophages has been reported in peritoneal fluid and ectopic endometriotic lesions; however, controversy exists regarding the composition and function of macrophage populations in eutopic endometrium of women with and without endometriosis. This study aimed to quantify macrophages in eutopic endometrium of women with and without endometriosis, during the early, mid and late proliferative and menstrual phases of the cycle. METHODS: Paraffin-embedded endometrial curettage blocks were selected from pathology archives. Seventy-six specimens from women with and without endometriosis were analysed using standard immunohistochemical techniques with CD68-PGM1 (phosphoglucomutase 1) clone antibody. Macrophages were counted according to their morphology over several fields of view. RESULTS: A significant increase in macrophage cell numbers was shown in eutopic endometrium in women with endometriosis (mean +/- SD, 182.7 +/- 72.9/mm(2)) during all stages of the proliferative phase compared with normal controls (101.6 +/- 53.4/mm(2); P < 0.001). Significant increase in macrophage density occurred in the control group during the mid-menstrual phase, Days 3-4 (P < 0.01), which was not observed in women with endometriosis. CONCLUSIONS: This study further supports an association between immune changes in eutopic endometrium and presence of endometriosis. However, it remains uncertain if eutopic immune changes are primary or secondary occurrences.