Annikki Liakka

Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Morphology and microsatellite instability in sporadic serrated and non-serrated colorectal cancer

Colorectal serrated adenocarcinoma originates from serrated adenoma, but definite histological criteria have not yet been established. It presents with frequent DNA microsatellite instability (MSI), but the frequency of low‐level (MSI‐L) and high‐level MSI (MSI‐H) and the expression of mismatch‐repair (MMR) enzymes in serrated adenocarcinoma are not known. To address these questions, morphological criteria for serrated cancers were established, their validity was tested, and MSI analysis was performed with NIH consensus markers and MMR enzyme immunohistochemistry for hMLH1, hMSH2, and hMSH6 in 35 serrated and 75 non‐serrated colorectal carcinomas. Serrated carcinomas frequently showed a serrated, mucinous or trabecular growth pattern; abundant eosinophilic cytoplasm; chromatin condensation; preserved polarity; and the absence of necrosis. With these features, it was possible to distinguish them from non‐serrated cancers, with the mean kappa score for five observers being 0.509. MSI analysis was successful in 31 serrated and 73 non‐serrated carcinomas. 54.8% of serrated carcinomas were microsatellite‐stable (MSS), 29.0% presented with MSI‐L, and 16.1% presented with MSI‐H, whereas 78.1% of non‐serrated carcinomas were MSS, 13.7% were MSI‐L, and 8.2% were MSI‐H. MSI‐L was more common in serrated cancers (p = 0.035) and it was associated with patchy immunohistochemical staining (33.3%) of MLH1. MSI‐H did not differ between serrated and non‐serrated cancers (p = 0.14). These results suggest that the biological background of serrated carcinomas differs from sporadic non‐serrated colorectal cancer, but is not directly related to MSI. Copyright

Postnatal Developmental Changes in the Pituitary-Ovarian Axis in Preterm and Term Infant Girls

CONTEXT Postnatal pituitary-testicular activation in infant boys is well characterized. However, the ovarian response to pituitary activation in infancy is less well understood. OBJECTIVE The aim of the study was to compare postnatal developmental changes in the pituitary-ovarian axis in preterm and term infant girls. PARTICIPANTS AND DESIGN Sixty-three infant girls, divided into three groups according to gestational age (GA) [i.e. full term (FT; n = 29; GA, 37-42 wk), near term (NT; n = 17; GA, 34-37 wk), and preterm (PT; n = 17; GA, 24-34 wk)] were examined monthly from 1 wk (D7) to 6 months (M1-M6) of age and reexamined at the corrected age of 14 months (cM14). MAIN OUTCOME MEASURES We performed a longitudinal follow-up of urinary FSH and serum anti-Müllerian hormone (AMH) levels and the number of follicles in transabdominal ovarian ultrasonography. RESULTS The postnatal FSH surge was stronger and more prolonged in NT and PT girls than in FT girls (P ≤ 0.001). Increased folliculogenesis and a rise in AMH levels were observed in all three groups after the FSH surge. In NT and PT girls, follicular development was delayed in comparison with FT girls, and a decrease in high FSH levels around the 40th postmenstrual week was temporally associated with the appearance of antral follicles in ultrasonography and an increase in AMH levels. CONCLUSIONS The postnatal FSH surge results in transient ovarian stimulation in term and preterm girls. A delay in ovarian folliculogenesis shown in ovarian ultrasonography and by low serum AMH levels may provide an explanation for the exaggerated FSH surge in NT and PT girls.

Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro

STUDY QUESTION Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? SUMMARY ANSWER In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. WHAT IS KNOWN ALREADY The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. STUDY DESIGN, SIZE, DURATION Prospective, university-based, case-control, in vitro study. PARTICIPANTS/MATERIALS, SETTING, METHODS Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 μM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models. WIDER IMPLICATIONS OF THE FINDINGS The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS. STUDY FUNDING/COMPETING INTERESTS Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests.

WNT4 is expressed in human fetal and adult ovaries and its signaling contributes to ovarian cell survival

WNT4 plays an important role in female sexual development and ovarian function. WNT4-deficiency leads disturbed development of the internal genitalia in mouse and human, and to a dramatic reduction of mouse oocytes. However, the expression and role of WNT4 in human ovaries is yet unknown. The expression of WNT4 mRNA and protein was studied in human adult and fetal ovaries (gestational ages 12-41 weeks), and the role of WNT4 in oocyte apoptosis was investigated in WNT4-deficient mice. WNT4 mRNA and protein were present in human ovaries throughout fetal development and in different follicular stages in adult ovaries. Compared with wild-type mice, WNT4-deficient mice had a markedly enhanced rate of oocyte apoptosis, with the highest values at gestational ages of 14.5 and 16.5 days post-coitum. The current results support a view that WNT4 may have a role in oocyte selection and follicle formation and maturation in human ovaries.

Differential expression of basement membrane components in lymphatic tissues

Peripheral lymphoid tissues act as important organs of immunological defense. Characteristic of their architecture is the rich reticular fiber meshwork composed of various extracellular matrix (ECM) molecules with which the stationary non-lymphatic cells stay in intimate contact and form channels through which the lymphatic cells travel. Here we studied the distribution of various laminin (Ln) chains and different types of collagens in human spleen, lymph node, and tonsil to clarify their chain-specific distribution. The most widely distributed proteins in all these organs were Ln chains α5, β1, γ1 and collagen types IV and XVIII, which were present in practically all compartments. Conversely, Ln α1, α2, α4, and type VII collagen showed a more restricted expression pattern. A unique feature was that Ln α3-, β3-, and γ2-chains, which normally are not localized to the vascular wall in non-lymphatic tissues, were present also in capillary basement membranes (BMs) of the follicular structures of lymph node and tonsil and in Ln α1-chain and type VII collagen also in the splenic white pulp. We also found that collagen XVII was exclusively present in the ring fibers of the spleen. The results indicate that BMs of lymphatic tissues contain a variety of macromolecules that probably contribute strongly to immunological events. In addition, capillaries of the lymphoid tissue exhibit a specified BM composition resembling that in epithelial BMs of non-lymphoid tissues.

Anti-Müllerian hormone as a predictor of follicular reserve in ovarian insufficiency: special emphasis on FSH-resistant ovaries

BACKGROUND Anti-Müllerian hormone (AMH) is secreted by ovarian granulosa cells and its serum levels reflect ovarian follicle reserve. The main objective of this study was to test the use of AMH assay in identifying women with primary amenorrhea (PA) and existing follicles and to study follicle phase dependent AMH secretion. METHODS Serum levels of AMH were measured in subjects with FSH-resistant ovaries (FSHRO, n= 12), primary ovarian insufficiency (POI) with PA (n= 11) or secondary amenorrhea (SA n= 20) of unknown etiology, and controls (n= 23), and in Turner syndrome (TS) [45,X (n= 18), mosaicism (n= 7), structural X chromosome abnormalities (SCA, n= 10)], and healthy controls (n= 34). RESULTS Serum levels of AMH in women with FSHRO were comparable with those in control women (2.76 ± 2.37 versus 3.77 ± 2.36 ng/ml) and significantly higher than in women with PA (0.05 ± 0.04 ng/ml; P < 0.001) or SA of unknown origin (0.12 ± 0.20 ng/ml; P < 0.001). TS girls/women with 45,X or SCA had low serum AMH levels (0.13 ± 0.09 and 0.27 ± 0.19 ng/ml) compared with their controls (3.34 ± 2.23 ng/ml) or subjects with mosaicism (2.33 ± 2.81 ng/ml). AMH expression was detected in granulosa cells of women with FSHRO but not in any of the 45,X fetal ovarian specimens. CONCLUSIONS A serum AMH assay could be used to identify patients with decreasing ovarian reserves and POI. Moreover, our results support the notion that AMH is secreted mainly by small non-selected follicles, since follicular granulosa cells were AMH-positive and serum AMH levels were normal/low normal in women with FSHRO, who lack follicle development beyond the small antral stage.

Distribution of laminin and types IV and III collagen in fetal, infant and adult human spleens

SummaryThe immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroths cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.

Feasibility of endometrial assessment after thermal ablation

OBJECTIVE To evaluate the feasibility of endometrial assessment after endometrial thermal ablation. STUDY DESIGN Prospective observational study. A total of 57 women (age 47-52 years), who had undergone endometrial thermal ablation as a treatment for heavy menstrual bleeding (HMB) 3-10 years (mean 6 years) earlier, were examined with transvaginal ultrasound and saline sonohysterography. Endometrial samples were collected with a Pipelle device. Visualisation of endometrium, access to uterine cavity, change in cavity length, success in outpatient endometrial sampling and success in sonohysterography were evaluated. RESULTS Endometrial thickness was 4.5mm in amenorrhoeic women (n=17), 5.6mm in eumenorrhoeic women (n=37) and 6.6mm in hypermenorrhoeic women (n=3). An endometrial sample was successfully taken in 44 (77%) women, and in 13 (23%) women endometrial sample taking failed. The length of the uterine cavity compared to the length measured before endometrial thermal ablation was 0.5-5 cm (mean 2 cm) shorter in 34 women, unchanged in four women and longer in five women. The uterine cavity distended regularly in only nine (16%) women. In 14 (25%) women the cavity distended irregularly or only partly, and in 24 (42%) women the uterine cavity did not distend at all, but appeared as a narrow tube. In 10 (18%) women the sonohysterography catheter did not enter the uterine cavity at all. CONCLUSION Endometrial assessment is compromised after previous endometrial thermal ablation. Both endometrial sampling and sonohysterography fail quite often, causing problems in diagnosis of abnormal bleeding. Intrauterine adhesions may also decrease the reliability of the endometrial sampling.

Regulation of Cell death in Human Fetal and Adult Ovaries - Role of Bok and Bcl-XL

Of eight million oocytes formed in fetal ovaries, only 400 are ovulated and the rest are degraded via apoptosis. Studies in rodents suggest an important role for Bok and Bcl-X(L) in ovarian apoptosis, but their expression patterns and roles in human ovaries are not well known. Protein expression of Bok and Bcl-X(L) as well as the death pathway effectors TNF and caspase-3 were determined in an important collection of samples consisting of human fetal and adult ovaries. A penetrant expression of Bok, Bcl-X(L), TNF and full length and cleaved caspase-3 were characterized in fetal ovaries, with specific patterns in oocytes and pre-granulosa/granulosa cells. Bok and Bcl-X(L) were detected also in adult ovaries. Lentiviral shRNA delivery demonstrated that loss of Bok markedly reduces vulnerability to apoptosis and, conversely, loss of Bcl-X(L) increases apoptosis in human granulosa tumour cell line. The results suggest important roles for Bok and Bcl-X(L) in human ovarian development, follicle maturation and apoptosis.