Molecular Human Reproduction - recent issues

Purification of germline stem cells from adult mammalian ovaries: a step closer towards control of the female biological clock?

Tilly, J. L., Telfer, E. E. — 2009-06-15

For decades it was believed that a non-renewable pool of oocyte-containing follicles is established in female mammals at birth. This cornerstone of reproductive biology was challenged 5 years ago by a study reporting on the presence of mitotically-active germ cells in juvenile and adult mouse ovaries. Additional findings presented in this study and others that followed further suggested that mammals retain the capacity to generate oocytes during adulthood; however, isolation of oocyte-producing germline stem cells (GSC) as unequivocal proof of their existence remained elusive. This piece of information now appears to have been provided by Ji Wu and colleagues. In addition to showing that proliferative germ cells resembling male spermatogonial stem cells can be purified from neonatal or adult mouse ovaries and maintained in vitro for months, transplantation studies demonstrated that these cells generate oocytes in ovaries of chemotherapy-sterilized recipients that fertilize and produce viable offspring. Although these findings do not establish that oogenesis occurs in adult females under physiological conditions, they strongly support the existence of GSC in adult mouse ovaries. If equivalent cells can be found in human ovaries, stem cell-based rejuvenation of the oocyte reserve in ovaries on the verge of failure may one day be realized.

The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption

Zhang, M., Ouyang, H., Xia, G. — 2009-06-15

Fully grown mammalian oocytes are arrested at the first meiotic prophase until a surge of gonadotrophin at the mid-cycle. The actions of gonadotrophins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), on oocyte meiotic resumption are believed to be mediated in large part through increasing the production of cyclic adenosine 3',5'-monophosphate and subsequent activation of mitogen-activated protein kinase (MAPK) in its surrounding cumulus granulosa cells. Recent findings indicate that gonadotrophins-induced epidermal growth factor-like growth factors, meiosis activating sterol and gonadal steroid hormones, possibly via protein kinase A II and protein kinase C pathways, are involved in the activation of MAPK. Another second messenger cyclic guanosine 3',5'-monophosphate induced by nitric oxide or natriuretic peptides system mediates the function of gonadotrophins during oocyte meiotic resumption. FSH and LH induced pathways may either directly overlap or each hormone may utilize redundant pathways in oocyte maturation. A detailed appreciation of different FSH and LH-activated signaling pathways in mammalian oocytes will be needed in understanding their actions in follicular development and oocyte maturation.

Superoxide dismutase expression in human cumulus oophorus cells

Matos, L., Stevenson, D., Gomes, F., Silva-Carvalho, J.L., Almeida, H. — 2009-06-15

Success in assisted reproductive techniques (ART) is influenced by gamete and embryo quality but the assessment of these parameters has been thwarted by the lack of reliable biomarkers. Follicular fluid and cumulus oophorus cells may provide biomarkers due to their close relationship to the oocyte. These cells produce antioxidants and thus protect the oocyte from oxidative damage exerted by reactive oxygen species (ROS). ROS and antioxidants are known to intervene in reproductive physiology and pathology, but their roles are unclear. It is hypothesized that superoxide dismutase (SOD), a first line antioxidant enzyme, is associated with oocyte quality. Cells obtained in the course of ART for the treatment of infertility due to male factor or female pathology were processed for SOD intracellular isoforms (CuZnSOD and MnSOD) immunodetection, total SOD activity and isoforms content. Cells presented strong positive staining for CuZnSOD and MnSOD. SOD activity decreased with increasing female age but was increased in endometriosis and in ovulatory dysfunction. When male factor was the cause for infertility, successful ART was associated with higher SOD activity. Variations in SOD emphasize the relevance of oxidative stress in the oocyte maturation process. These variations also suggest that SOD is a potential biomarker for ART success.

The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice

2009-06-15

The present study investigated the role of the N, N'-dimethylbiguanide metformin (50 mg/kg body weight in 0.05 ml water, given orally with a canulla) in preventing the adverse effects generated by hyperandrogenism on uterine function. Daily injection of dehydroepiandrosterone (DHEA: 6 mg/100 g body weight in 0.1 ml oil) for 20 consecutive days induces polycystic ovaries in BALB/c mice. In this model we found that DHEA produced alterations on uterine histology closely related to the development of pre-cancerous structures concomitantly with increased incidence of uterine apoptosis. The injection of DHEA induced a pro-inflammatory status since uterine prostaglandin (PG) F2 alpha levels and cyclooxygenase 2 were increased although PGE levels were decreased. Furthermore, DHEA promoted a pro-oxidant status since it increased nitric oxide synthase (NOS) activity and decreased superoxide dismutase and catalase activities and the antioxidant metabolite glutathione levels. DHEA also regulated the percentages of CD4+ and CD8+ T lymphocyte that infiltrate uterine tissue. When metformin was administered together with DHEA uterine histology and apoptosis did not differ when compared with controls. Therefore, metformin prevented the pro-inflammatory and pro-oxidative status generated by DHEA and restores the ratios of CD4+ and CD8+ T cells to those observed in controls. We conclude that metformin is able to restore either directly or indirectly uterine function by preventing some inflammatory and oxidative alterations produced by hyperandrogenism.

Protein processing by the placental protease, cathepsin P

Hassanein, M., Bojja, A. S., Glazewski, L., Lu, G., Mason, R.W. — 2009-06-15

Cathepsin P is a member of a family of placentally expressed cathepsins (PECs). The closest human homolog of cathepsin P is cathepsin L, a broad specificity enzyme that has functions in many tissues in addition to placenta. The gene duplications that gave rise to the PECs provide a rare opportunity to define proteolytic functions in placenta, a transient organ unique to mammals. Peptidyl substrate and inhibitor libraries have shown that cathepsin P has evolved an unusually restricted preference for substrates containing hydrophobic amino acids. Proteomic techniques were used to probe for substrates of this enzyme. Recombinant cathepsin P was incubated with rat choriocarcinoma (Rcho-1) cell proteins to identify substrates using two-dimensional difference gel electrophoresis. Substrate proteins were excised from gels and characterized by trypsin digestion and MALDI MS/MS. Two endoplasmic reticulum (ER) proteins, gp96 and calreticulin, emerged as potential substrates, and western blotting showed that these proteins are processed by cathepsin P from their C-terminus, removing the KDEL ER retention signal. Immunohistochemistry showed that a portion of cathepsin P co-localizes with calreticulin in Rcho-1 cells. Extracellular calreticulin induces differentiation of Rcho-1 cells, indicating a potential role of cathepsin P in processing and secretion of calreticulin during differentiation of trophoblast giant cells.

ROCK2 allelic variants are not associated with pre-eclampsia susceptibility in the Finnish population

2009-06-15

The rho-associated coiled-coil protein kinase 2 (ROCK2) gene has been suggested to associate with general hypertension and is therefore a plausible functional candidate gene for pre-eclampsia. ROCK2 maps to chromosome 2p25, which we have implicated previously in a linkage study of pre-eclampsia. We have re-sequenced exons and putative promoter region of ROCK2 in up to 30 pre-eclampsia patients and 22 controls and genotyped putative functional single-nucleotide polymorphisms (SNPs) as well as tagging SNPs from HapMap in a Finnish case–control data set—340 affected and 357 matched control individuals—for a genetic association study of ROCK2 in pre-eclampsia. Even though several new SNPs were discovered, we did not detect significant allelic or haplotypic association between ROCK2 and pre-eclampsia. We assessed ROCK2 expression in placentas by microarray analysis, but no significant expression differences were observed when comparing preeclamptic and normotensive pregnancies. We conclude that common genetic variation in ROCK2 is unlikely to make a major contribution to the risk of pre-eclampsia, but cannot exclude the possibility of having missed non-coding functional variants or rare coding variants.

Thrombin and interleukin-1{beta} decrease HOX gene expression in human first trimester decidual cells: implications for pregnancy loss

Sarno, J., Schatz, F., Huang, S. J., Lockwood, C., Taylor, H. S. — 2009-06-15

Bleeding or inflammation in early pregnancy may result in pregnancy loss or defective implantation. Their effect on HOX gene expression in first trimester decidua is unknown. Bleeding results in thrombin generation, although infection or inflammation results in production of cytokines typified by Interleukin-1β (IL-1β). First trimester decidual cells were pretreated with 17β estradiol (E₂), medroxyprogesterone acetate (MPA) or both and subsequently treated with thrombin or IL-1β. Affymetrix microarray analysis was used to assess the expression of all HOX genes and confirmed using real-time RT–PCR. E₂ or MPA treatment resulted in significant increases in HOXA10 and HOXA11. Subsequent treatment with thrombin resulted in diminished expression of HOXA10 and HOXA9. Treatment with IL-1β resulted in decreased expression of HOXA1, 3, 9, 10 and 11. HOXA10 expression was reduced by 70% after thrombin treatment (P = 0.018) and by 90% after IL-1β treatment (P = 0.004). HOXA11 mRNA expression was decreased by 88% after IL-1β treatment (P < 0.001), but not by thrombin treatment. Decidua was collected at the time of elective termination of pregnancy (n = 10) or surgical treatment of spontaneous pregnancy loss (n = 10). Real-time PCR and western analysis demonstrated decreased HOXA10 and HOXA11 RNA and protein expression in the decidua of spontaneous pregnancy loss compared with that of viable pregnancies. In conclusion, multiple HOX genes are expressed in decidual cells and inhibited by thrombin and IL-1β. Since HOXA10 and HOXA11 are known to be necessary for successful pregnancy, these findings suggest a molecular mechanism by which bleeding or inflammation may affect pregnancy outcome.

Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives

Manna, P. R., Dyson, M. T., Stocco, D. M. — 2009-05-04

Steroid hormones are synthesized in the adrenal gland, gonads, placenta and brain and are critical for normal reproductive function and bodily homeostasis. The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroid biosynthesis, i.e. the delivery of cholesterol from the outer to the inner mitochondrial membrane. The expression of the StAR protein is predominantly regulated by cAMP-dependent mechanisms in the adrenal and gonads. Whereas StAR plays an indispensable role in the regulation of steroid biosynthesis, a complete understanding of the regulation of its expression and function in steroidogenesis is not available. It has become clear that the regulation of StAR gene expression is a complex process that involves the interaction of a diversity of hormones and multiple signaling pathways that coordinate the cooperation and interaction of transcriptional machinery, as well as a number of post-transcriptional mechanisms that govern mRNA and protein expression. However, information is lacking on how the StAR gene is regulated in vivo such that it is expressed at appropriate times during development and is confined to the steroidogenic cells. Thus, it is not surprising that the precise mechanism involved in the regulation of StAR gene has not yet been established, which is the key to understanding the regulation of steroidogenesis in the context of both male and female development and function.

Do circulating blood cells contribute to maternal tissue remodeling and embryo-maternal cross-talk around the implantation period?

Fujiwara, H. — 2009-05-04

In early pregnancy, human chorionic gonadotrophin (HCG) stimulates the corpus luteum (CL) of pregnancy to produce progesterone, which in turn maintains human embryo implantation in the uterus. In addition to this embryo–maternal cross-talk via the endocrine systems through blood circulation, accumulating evidence suggests that circulating blood cells also play an important role in embryo implantation. Peripheral blood mononuclear cells (PBMC) derived from pregnant women increased the progesterone production by luteal cells and promoted the invasion of embryos in vitro. Recombinant-HCG increased chemokine production by PBMC through lectin–glycan interaction and enhanced the effects of PBMC on embryo invasion. Later, it was shown that not only PBMC, but also circulating platelets were possible sources of these chemokines that promote extravillous trophoblast invasion to reconstruct maternal endometrial artery. Circulating platelets were also proposed to induce neovascularization during CL formation. Furthermore, intrauterine administration of autologous PBMC effectively improved live birth, pregnancy and implantation rates in patients with repeated (four or more) implantation failures during in vitro fertilization therapy. These findings suggest that circulating blood cells positively contribute to maternal tissue remodeling and embryo–maternal cross-talk around the implantation period in cooperation with the endocrine system.

Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells

2009-05-04

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.

In-vivo gene transfer induces transgene expression in cells and secretions of the mouse cauda epididymis

Esponda, P., Carballada, R. — 2009-05-04

Mouse cauda epididymis were in-vivo transfected using the lipid FuGENE 6 as gene vector. Two gene constructions were employed: the p-GeneGRIP which codifies for the Green Fluorescent Protein (GFP) and the pSEAP-control that expresses an alkaline phosphatase as a secretion. Transfection was detected by fluorescence and appeared in the nucleus and cytoplasm of epithelial cells. Transfection was observed in 39.70% of cells after 2 days and in 31.77% after 7 days, and then diminished progressively. Moreover, the presence of the transgene in the DNA isolated from treated epididymides was observed by polymerase chain reaction. GFP gene expression appeared in large areas of the cauda epididymis and it was observed exclusively in the cytoplasm of epithelial cells. GFP gene expression occurred during 2 weeks after gene injection and occupied 32.24, 29.98 and 22.37% of the area of the tubules when analyzed 2, 7 and 15 days after gene injection. The cauda was also analyzed in toto and showed similar results. The use of the pSEAP-control gene showed that cauda epididymis secretions can also be modified by the transfection procedure. A significant increase of alkaline phosphatase activity appeared in the epididymal fluids 7 days after gene injection. These results indicate that transfection procedures could be an important tool in the future to study epididymal physiology or to change the fertilizing ability of spermatozoa.

A molecular evaluation of germ cell death induced by etoposide in pubertal rat testes

Ortiz, R. J., Lizama, C., Codelia, V. A., Moreno, R. D. — 2009-05-04

Etoposide is widely used in the treatment of patients with testicular cancer. The mechanism underlying apoptosis induction in cancer cells has been studied in different cell types, but it is not known whether the same factors participate in viable germ cells undergoing programmed cell death. Since testicular cancer primarily affects young males, we used pubertal rats (21 days old) as a model to determine different apoptotic parameters after etoposide treatment in healthy testes. We found that one intratesticular injection of etoposide (1.2 µg/testis) induced a significant increase in spermatocytes undergoing apoptosis, along with activation of caspase-9, -8 and -3 after 24 h of treatment. Spermatocyte apoptosis was inhibited when a general caspase inhibitor was added along with etoposide. Etoposide induces a significant stabilization/activation of p53, resulting in an increase level of this protein. The mRNA of Bcl-2 antagonist of cell death (BAD), a pro-apoptotic gene and a transcriptional target of p53, was significantly increased after etoposide treatment. Thus, our results suggest a single injection of etoposide induces apoptosis in healthy pachytene spermatocytes mediated by p53 and caspase activation. These findings will assist the search for new therapies to prevent the deleterious effect of cancer drugs upon normal cells.

Mass spectrometry analysis of dynamic post-translational modifications of TH2B during spermatogenesis

Lu, S., Xie, Y. M., Li, X., Luo, J., Shi, X. Q., Hong, X., Pan, Y. H., Ma, X. — 2009-05-04

TH2B, an important testis histone, plays a key role in remodeling chromatin structure during spermatogenesis. We present a detailed study of post-translational modifications (PTMs) of histone TH2B from different developmental stages of sperm cells, using a combination of high performance liquid chromatography, enzymatic Glu-c digestions of peptides, liquid chromatography–mass spectrometry (LC–MS) and LC–MS/MS analysis. The results showed modification patterns of the intact histone TH2B during spermatogenesis. Acetylated TH2B was most abundant in spermatogonia (28.9%) when compared with the spermatocytes (8.3%) and round spermatids (11.2%). Several new PTMs of TH2B were identified. In spermatogonia, spermatocytes and round spermatids, T116 and K117, were modified by phosphorylation and methylation, respectively, forming a novel ‘phospho switch’ site. The identified modification patterns of histone TH2B in spermatogenic cells provides a basis for future studies on histone coding and epigenetic regulation during spermatogenesis.

Regulation of 3{beta}-hydroxysteroid dehydrogenase type 1 and type 2 gene expression and function in the human ovarian surface epithelium by cytokines

2009-05-04

The human ovarian surface epithelium (hOSE) is a squamous-to-cuboidal layer that surrounds the ovary. hOSE undergoes injury and repair cycles as a result of ovulation-induced inflammation, an event relevant to the development of epithelial ovarian cancer (EOC). Locally produced steroids mediate the response to inflammation. 3β-Hydroxysteroid dehydrogenase (3β-HSD) drives the intracrine generation of progestogens and androgens that potentially affect cell survival and proliferation. We therefore investigated the regulation of 3β-HSD along with downstream steroid signalling in hOSE. Double immunofluorescence of cultured primary hOSE cells confirmed the expression of 3β-HSD protein Interleukin (IL). IL-1 treatment of primary cells to mimic ovulation-associated inflammation suppressed 3β-HSD1 expression and stimulated 3β-HSD2 mRNA (P < 0.001), without affecting total 3β-HSD protein and activity or androgen or progesterone receptor (PR) mRNA levels. Conversely, IL-4 as a proxy for a post-ovulatory healing cytokine increased both 3β-HSD transcripts, total protein and activity (P < 0.01). IL-4 also suppressed androgen receptor expression (P < 0.01) without affecting that of the PR, thereby potentially sustaining both progesterone biosynthesis and its underlying signalling in the ovarian surface. 3β-HSD protein was immunodetectable in primary ascites of women who were diagnosed with EOC but both mRNA transcripts were diminished relative to normal cells (P < 0.05). Notably, this difference was countered by IL-4 treatment (P < 0.01). We conclude that stimulation by IL-4 could be physiologically relevant to post-ovulatory ovarian healing and suggest a novel therapeutic strategy for the activation of progesterone-associated apoptosis in ovarian cancer. Also, our results suggest an attenuation of 3β-HSD expression in EOC although further studies are required for confirmation.

OCT4B1 isoform: the novel OCT4 alternative spliced variant as a putative marker of stemness

2009-04-07

A novel OCT4 alternative spliced variant (OCT 4B1) is deduced to be the isoform present in 59 hESC lines characterised by the International Stem Cell Initiative (ISCI) rather than OCT4A as previously assumed. The new variant may be a more reliable marker of stemness than OCT4A and studies are needed to test this.

The role of proteomics in defining the human embryonic secretome

Katz-Jaffe, M.G., McReynolds, S., Gardner, D.K., Schoolcraft, W.B. — 2009-04-07

Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers.

Cryopreservation of porcine oocytes: recent advances

Zhou, G.-B., Li, N. — 2009-04-07

Successful cryopreservation of porcine gametes and embryos has been very challenging due to their sensitivity to cryoinjuries. Although considerable improvements have been achieved in the vitrification of porcine embryos, there has been no offspring born from the vitrified oocytes in this species. Porcine oocytes characteristically contain large amounts of cytoplasmic lipids that are major obstacles limiting efficient cryopreservation. These droplets together with structures such as mitochondria, membranes, cortical granules and basic components of the spindle and cytoskeleton (microtubules and microfilaments) often incur serious damage during cooling and warming. According to recent reports, the proper combinations of permeable and non-permeable cryoprotectants and vitrification with high cooling and warming rates may increase the survival of porcine oocytes. The cryotolerance of porcine oocytes may also be enhanced by removal of the chilling-sensitive lipid droplets, supplementation of cytoskeleton relaxants in vitrification solutions, or high hydrostatic pressure pretreatment of oocytes before cryopreservation. The improvement in cryopreservation methodology for porcine oocytes will no doubt augment other technologies such as pig cloning and the establishment of a gene bank for transgenic pigs.

Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy

2009-04-07

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT–PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic location of the trophoblast.

Do mitochondrial mutations cause recurrent miscarriage?

2009-04-07

The cause of recurrent miscarriage (RM) can be identified in ~50% of cases, whereas in others, unknown genetic factors are actively being sought. As mitochondrial functions, and therefore also the mitochondrial genome [mitochondrial DNA (mtDNA)], have an important role in human development, through ATP production and participation in apoptosis, we aimed to study the role of mtDNA variations in RM. We screened 48 women with RM and 48 age-matched control women for heteroplasmic mitochondrial mutations using denaturing high performance liquid chromatography, a sensitive method that can detect ~5% heteroplasmy. As a result, we detected a heteroplasmic mtDNA variation in 13 RM women (27%) and in 9 control women (19%). Seven synonymous and five non-synonymous changes were detected within coding regions. In addition, seven heteroplasmic variations were detected within the non-coding control region. We were also able to show the presence of the variations in eight placental samples from three heteroplasmic women. In three of these cases, the proportion of variant mtDNA was higher in the placenta compared with that in the mother. We conclude that our sensitive methodology revealed a higher frequency of samples with heteroplasmic variations than expected in women with both RM and controls. However, no apparent increased frequency of heteroplasmic mtDNA variations or amounts of aberrant mtDNA was detected in the RM group. In addition, none of the detected variations were previously known to be pathogenic and therefore they are an unlikely cause of miscarriage.

Altered activity of lysophospholipase D, which produces bioactive lysophosphatidic acid and choline, in serum from women with pathological pregnancy

Tokumura, A., Kume, T., Taira, S., Yasuda, K., Kanzaki, H. — 2009-04-07

Altered lipid metabolism is associated with human abnormal pregnancy, such as pre-eclampsia and preterm labor, and potentially leads to fetus loss. A causative factor for the onset and progress of the systemic multifactorial syndromes associated with the pathological pregnancy is oxidized low-density lipoprotein, an active identity of which was postulated to be lysophosphatidic acid (LPA). We previously found that LPA is produced extracellularly by plasma lysophospholipase D (lysoPLD) activity of autotaxin, a tumor cell motility-stimulating protein. In this study, a convenient assay based on the choline released from endogenous substrate or exogenous lysophosphatidylcholine (LPC) was used for comparison of serum lysoPLD activity among patients with normal and abnormal pregnancy. The serum choline-producing activity was found to be mainly due to autotaxin, and dependent on its dilution rate. There was some association between low dilution dependency of serum lysoPLD activity toward an exogenous LPC and high lysoPLD activity toward endogenous substrates in cases of patients with preterm labor and pre-eclampsia. However, there was no difference in the serum level of LPC between women with normal pregnancy and those with pathological pregnancy. These results indicate that production of bioactive LPA by lysoPLD activity is elevated by an unknown mechanism that may be related to increased availability of endogenous substrates LPC, but not its concentration in human serum. If the level of LPA in blood circulation is elevated in the pathological pregnancies in vivo, it may play a role in induction and/or progression of systemic vascular dysfunction seen patients with preterm labor or pre-eclampsia.

Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation

2009-04-07

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs), present in epithelial cells, monocytes and dendritic cells. This study details their expression, regulation and role in human endometrium. Real-time PCR showed that NOD1 mRNA is constitutively expressed in endometrium. NOD2 is up-regulated in the late secretory phase of the menstrual cycle suggesting a role in menstruation. Both proteins are immunolocalized in endometrial epithelium, stroma and endothelium. In first trimester, decidua NODs are present in decidualized stroma. NOD function was examined in endometrial stromal cells (ESCs) and endometrial epithelial cells (EEpCs) in vitro. IB is up-regulated by stimulation of ESC and EEpC with an NOD1 ligand. IB, IL-8 and TNF mRNA expression is increased in EEpC by a NOD2 ligand. NOD2 mRNA expression increases in response to IL-1 treatment while NOD1 transcripts are unaltered. NOD1 mRNA is increased in an in vitro model of decidualization of ESC. In summary, we report expression of NOD1 and NOD2 in human endometrium and show that they are differentially regulated. NOD2 and, to a lesser extent, NOD1 can function to increase expression of innate immune molecules in endometrium. NODs may have a role in innate immune protection in the uterus and NOD2 may regulate inflammation associated with menstruation.