Global Transcriptomic Analysis of Placentas from Women with Gestational SARS-CoV-2 Infection during the Third Trimester of Pregnancy.

The COVID-19 pandemic has had a significant and enduring influence on global health, including maternal and fetal well-being. Evidence suggests that placental dysfunction is a potential consequence of SARS-CoV-2 infection during pregnancy, which may result in adverse outcomes such as preeclampsia and preterm birth. However, the molecular mechanisms underlying this association remain unclear, and it is uncertain whether a mature placenta can protect the fetus from SARS-CoV-2 infection. To address the above gap, we conducted a transcriptome-based study of the placenta in both maternal and fetal compartments. We collected placental samples from 16 women immediately after term delivery, seven of which had SARS-CoV-2 infection confirmed by PCR before parturition. Notably, we did not detect any viral load in either the maternal or fetal compartments of the placenta, regardless of symptomatic status. We separately extracted total RNA from placental tissues from maternal and fetal compartments, constructed cDNA libraries, and sequenced them to assess mRNA. Our analysis revealed 635 differentially expressed genes when a false discovery rate (FDR ≤ 0.05) was applied in the maternal placental tissue, with 518 upregulated and 117 downregulated genes in the SARS-CoV-2-positive women (n = 6) compared with the healthy SARS-CoV-2-negative women (n = 8). In contrast, the fetal compartment did not exhibit any significant changes in gene expression with SARS-CoV-2 infection. We observed a significant downregulation of nine genes belonging to the pregnancy-specific glycoprotein related to the immunoglobulin superfamily in the maternal compartment with active SARS-CoV-2 infection (fold change range from -13.70 to -5.28; FDR ≤ 0.01). Additionally, comparing symptomatic women with healthy women, we identified 1788 DEGs. Furthermore, a signaling pathway enrichment analysis revealed that pathways related to oxidative phosphorylation, insulin secretion, cortisol synthesis, estrogen signaling, oxytocin signaling, antigen processing, and presentation were altered significantly in symptomatic women. Overall, our study sheds light on the molecular mechanisms underlying the reported clinical risks of preeclampsia and preterm delivery in women with SARS-CoV-2 infection. Nonetheless, studies with larger sample sizes are warranted to further deepen our understanding of the molecular mechanisms of the placenta's anti-viral effects in maternal SARS-CoV-2 infection.

Bromocriptine inhibits proliferation in the endometrium from women with adenomyosis.

Objective: Bromocriptine treatment has been shown to reduce menstrual bleeding and pain in women with adenomyosis in a pilot clinical trial. The underlying mechanism contributing to the treatment effect is however unknown. The purpose of this study was to explore the effect of bromocriptine on the proliferation and migration properties of the endometrium in women with adenomyosis, by assessing cellular and molecular changes after six months of vaginal bromocriptine treatment. Methods: Endometrial specimens were collected during the proliferative phase from women with adenomyosis (n=6) before (baseline) and after six months of treatment with vaginal bromocriptine. Immunohistochemistry was used to determine changes in the protein expression of Ki67 in the endometrium of women with adenomyosis. Primary endometrial stromal cells isolated at baseline were expanded in vitro and exposed to different doses of bromocriptine to determine the optimal half-maximum inhibitory concentration (IC50) using CellTiter-Blue® Cell Viability Assay. Cell proliferation was assessed by bromodeoxyuridine ELISA assay and Ki67 gene expression was checked by real-time PCR. The migratory ability of endometrial stromal cells was determined by wound healing and transwell migration assays. Small RNA sequencing was applied on tissues collected from women with adenomyosis before and after bromocriptine treatment to identify differentially expressed microRNAs (miRNAs) after bromocriptine treatment. Bioinformatic methods were used for target gene prediction and the identification of biological pathways by enrichment procedures. Results: Vaginal bromocriptine treatment reduced the Ki67 protein expression in the endometrium of women with adenomyosis and did not change the prolactin mRNA expression and protein concentration of prolactin in endometrial tissues. Bromocriptine significantly inhibited the proliferative and migrative abilities of endometrial stromal cells derived from women with adenomyosis in vitro. Moreover, small RNA sequencing revealed 27 differentially expressed miRNAs between the endometrium of women with adenomyosis before and after six months of vaginal bromocriptine treatment. KEGG pathway analysis on targeted genes of 27 miRNAs showed that several signaling pathways associated with cell proliferation and apoptosis were enriched after bromocriptine treatment. Conclusion: Bromocriptine treatment exhibits an anti-proliferative effect in the endometrium of women with adenomyosis in vivo and in vitro. Bromocriptine might inhibit the proliferation of endometrial tissue in adenomyosis in part through the regulation of dysregulated microRNAs and proliferation-associated signaling pathways.

Effects of Estradiol/Micronized Progesterone vs. Conjugated Equine Estrogens/Medroxyprogesterone Acetate on Breast Cancer Gene Expression in Healthy Postmenopausal Women.

Recent studies suggest estradiol (E2)/natural progesterone (P) confers less breast cancer risk compared with conjugated equine estrogens (CEE)/synthetic progestogens. We investigate if differences in the regulation of breast cancer-related gene expression could provide some explanation. This study is a subset of a monocentric, 2-way, open observer-blinded, phase 4 randomized controlled trial on healthy postmenopausal women with climacteric symptoms (ClinicalTrials.gov; EUCTR-2005/001016-51). Study medication was two 28-day cycles of sequential hormone treatment with oral 0.625 mg CEE and 5 mg of oral medroxyprogesterone acetate (MPA) or 1.5 mg E2 as percutaneous gel/day with the addition of 200 mg oral micronized P. MPA and P were added days 15-28/cycle. Material from two core-needle breast biopsies in 15 women in each group was subject to quantitative PCR (Q-PCR). The primary endpoint was a change in breast carcinoma development gene expression. In the first eight consecutive women, RNA was extracted at baseline and after two months of treatment and subjected to microarray for 28856 genes and Ingenuity Pathways Analysis (IPA) to identify risk factor genes. Microarray analysis showed 3272 genes regulated with a fold-change of >±1.4. IPA showed 225 genes belonging to mammary-tumor development function: 198 for CEE/MPA vs. 34 for E2/P. Sixteen genes involved in mammary tumor inclination were subject to Q-PCR, inclining the CEE/MPA group towards an increased risk for breast carcinoma compared to the E2/P group at a very high significance level (p = 3.1 × 10-8, z-score 1.94). The combination of E2/P affected breast cancer-related genes much less than CEE/MPA.

Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.

STUDY QUESTION: Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells? SUMMARY ANSWER: A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted. WHAT IS KNOWN ALREADY: The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted. STUDY DESIGN SIZE DURATION: Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in the embryo implantation process were highlighted. LARGE SCALE DATA: RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929. LIMITATIONS REASONS FOR CAUTION: Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only. WIDER IMPLICATIONS OF THE FINDINGS: The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest.

Uterine fluid microRNAs are dysregulated in women with recurrent implantation failure.

STUDY QUESTION: Is the composition of microRNAs (miRNAs) in uterine fluid (UF) of women with recurrent implantation failure (RIF) different from that of healthy fertile women? SUMMARY ANSWER: The composition of miRNAs in UF of women with RIF is different from that of healthy fertile women and the dysregulated miRNAs are associated with impaired endometrial receptivity and embryo implantation. WHAT IS KNOWN ALREADY: It has previously been demonstrated that the miRNAs secreted from endometrial cells into the UF contribute to the achievement of endometrial receptivity. Endometrial miRNAs are dysregulated in women with RIF. STUDY DESIGN, SIZE, DURATION: In this descriptive laboratory case-control study, miRNA abundancy was compared between UF collected during implantation phase from healthy fertile women (n = 17) and women with RIF (n = 34), which was defined as three failed IVF cycles with high-quality embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recruitment of study subjects and sampling of UF were performed at two university clinics in Stockholm, Sweden and Tartu, Estonia. The study participants monitored their menstrual cycles using an LH test kit. The UF samples were collected on Day LH + 7-9 by flushing with saline. Samples were processed for small RNA sequencing and mapped for miRNAs. The differential abundance of miRNAs in UF was compared between the two groups using differential expression analysis (DESeq2). Further downstream analyses, including miRNA target gene prediction (miRTarBase), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (g:Profiler) and external validation using relevant published data, were performed on the dysregulated miRNAs. Two miRNAs were technically validated with quantitative real-time PCR (RT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE: After processing of the sequencing data, there were 15 samples in the healthy fertile group and 33 samples in the RIF group. We found 61 differentially abundant UF miRNAs (34 upregulated and 27 downregulated) in RIF compared to healthy women with a false discovery rate of <0.05 and a fold change (FC) of ≤-2 or ≥2. When analyzed with published literature, we found that several of the differentially abundant miRNAs are expressed in endometrial epithelial cells and have been reported in endometrial extracellular vesicles and in association with endometrial receptivity and RIF. Their predicted target genes were further expressed both in the trophectodermal cells of blastocyst-stage embryos and endometrial mid-secretory epithelial cells, as assessed by publicly available single-cell transcriptome-sequencing studies. Pathway analysis further revealed that 25 pathways, having key roles in endometrial receptivity and implantation, were significantly enriched. Hsa-miR-486-5p (FC -20.32; P-value = 0.004) and hsa-miR-92b-3p (FC -9.72; P-value = 0.004) were successfully technically validated with RT-PCR. LARGE SCALE DATA: The data are available in Gene Expression Omnibus (GEO) at https://www.ncbi.nlm.nih.gov/geo/ with GEO accession number: GSE173289. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study with a limited number of study participants. Moreover, the identified differentially abundant miRNAs should be validated in a larger study cohort, and the predicted miRNA target genes and enriched pathways in RIF need to be confirmed and further explored in vitro. WIDER IMPLICATIONS OF THE FINDINGS: RIF is a major challenge in the current IVF setting with no diagnostic markers nor effective treatment options at hand. For the first time, total miRNAs have been extensively mapped in receptive phase UF of both healthy women with proven fertility and women diagnosed with RIF. Our observations shed further light on the molecular mechanisms behind RIF, with possible implications in future biomarker and clinical treatment studies. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the Swedish Research Council (2017-00932), a joint grant from Region Stockholm and Karolinska Institutet (ALF Medicine 2020, FoUI-954072), Estonian Research Council (PRG1076), Horizon 2020 innovation (ERIN, EU952516) and European Commission and Enterprise Estonia (EU48695). The authors have no competing interests to declare for the current study.

Syndecan-1 modulates the invasive potential of endometrioma via TGF-ß signalling in a subgroup of women with endometriosis.

STUDY QUESTION: What is the physiological role of transforming growth factor-beta (TGF-ß1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis? SUMMARY ANSWER: We observed an abnormal, pro-invasive phenotype in a subgroup of samples with ovarian endometriosis, which was reversed by combining gene silencing of SDC1 with the TGF-ß1 treatment. WHAT IS KNOWN ALREADY: Women with endometriosis express high levels of TGF-ß1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-ß1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity. STUDY DESIGN, SIZE, DURATION: We utilized a pre-validated panel of stem- and cancer cell-associated markers on endometriotic tissue (n = 15) to stratify subgroups of women with endometriosis. Furthermore, CD90+CD73+CD105+ (SC+) endometriotic stromal cells from these patient subgroups were explored for their invasive behaviour in vitro by transient gene inhibition of SDC1 or SDC4, both in the presence or absence of TGF-ß1 treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometriotic cyst biopsies (n = 15) were obtained from women diagnosed with ovarian endometriosis (ASRM Stage III-IV). Gene expression variability was assessed on tissue samples by applying gene clustering tools for the dataset generated from the pre-validated panel of markers. Three-dimensional (3D) spheroids from endometriotic SC+ were treated in vitro with increasing doses of TGF-ß1 or the TGFBRI/II inhibitor Ly2109761 and assessed for SDC1, SDC4 expression and in vitro 3D-spheroid invasion. Transcriptomic signatures from the invaded 3D spheroids were evaluated upon combining transient gene silencing of SDC1 or SDC4, both in presence or absence of TGF-ß1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-ß1 or TGFBRI/II inhibitor using atomic force microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Gene clustering analysis revealed that endometriotic tissues displayed variability in their gene expression patterns; a small subgroup of samples (2/15, Endo-hi) exhibited high levels of SDC1, SDC4 and molecules involved in TGF-ß signalling (TGF-ß1, ESR1, CTNNB1, SNAI1, BMI1). The remaining endometriotic samples (Endo-lo) showed a uniform, low gene expression profile. Three-dimensional spheroids derived from Endo-hi SC+ but not Endo-lo SC+ samples showed an aberrant expression of SDC1 and exhibited enhanced 3D-spheroid invasion in vitro, upon rhTGF-ß1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-ß1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-ß1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling. LARGE SCALE DATA: Transcriptomic data were deposited in NCBI's Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122. LIMITATIONS, REASONS FOR CAUTION: It is estimated that about 2.5% of endometriosis patients have a potential risk for developing ovarian cancer later in life. It is possible that the pro-oncogenic molecular changes observed in this cohort of endometriotic samples may not correlate with clinical occurrence of ovarian cancer later in life, thus a validation will be required. WIDER IMPLICATIONS OF THE FINDINGS: This study emphasizes the importance of interactions between syndecans and TGF-ß1 in the pathophysiology of endometriosis. We believe that this knowledge could be important in order to better understand endometriosis-associated complications such as ovarian cancer or infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Cancerfonden (CAN 2016/696), Radiumhemmets Forskningsfonder (Project no. 154143 and 184033), EU MSCA-RISE-2015 project MOMENDO (691058), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695) and Karolinska Institute. Authors do not have any conflict of interest.

Uterine Fluid Proteins for Minimally Invasive Assessment of Endometrial Receptivity.

CONTEXT: Clinically used endometrial (EM) receptivity assays are based on transcriptomic patterning of biopsies at midsecretory endometrium (MSE) to identify the possible displacement or disruption of window of implantation (WOI) in patients with recurrent implantation failure (RIF). However, biopsies are invasive and cannot be performed in the same cycle with in vitro fertilization embryo transfer, while uterine fluid (UF) analysis is considered minimally invasive and can immediately precede embryo transfer. OBJECTIVE: To determine whether UF proteome can be used for WOI monitoring and whether it would highlight the etiology of RIF. PATIENTS: Paired early secretory endometrial (ESE) and MSE UF samples from six fertile control women for discovery, and an additional 11 paired ESE/MSE samples from controls and 29 MSE samples from RIF patients for validation. RESULTS: Using discovery mass spectrometry (MS) proteomics we detected 3158 proteins from secretory phase UF of which 367 undergo significant (q < 0.05) proteomic changes while transitioning from ESE to MSE. Forty-five proteins were further validated with targeted MS, and 21 were found to display similar levels between control ESE and RIF MSE, indicating displacement of the WOI. A panel of PGR, NNMT, SLC26A2 and LCN2 demonstrated specificity and sensitivity of 91.7% for distinguishing MSE from ESE samples. The same panel distinguished control MSE samples from RIF MSE with a 91.7% specificity and 96.6% sensitivity. CONCLUSION: UF proteins can be used for estimating uterine receptivity with minimal invasiveness. Women with RIF appear to have altered MSE UF profiles that may contribute to their low IVF success rate.

Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis.

STUDY QUESTION: Is there molecular evidence for a link between endometriosis and endometriosis-associated ovarian cancers (EAOC)? STUDY ANSWER: We identified aberrant gene expression signatures associated with malignant transformation in a small subgroup of women with ovarian endometriosis. WHAT IS KNOWN ALREADY: Epidemiological studies have shown an increased risk of EAOC in women with ovarian endometriosis. However, the cellular and molecular changes leading to EAOC are largely unexplored. STUDY DESIGN, SIZE, DURATION: CD73+CD90+CD105+ multipotent stem cells/progenitors (SC cohort) were isolated from endometrium (n = 18) and endometrioma (n = 11) of endometriosis patients as well as from the endometrium of healthy women (n = 14). Extensive phenotypic and functional analyses were performed in vitro on expanded multipotent stem cells/progenitors to confirm their altered characteristics. Aberrant gene signatures were also validated in paired-endometrium and -endometrioma tissue samples from another cohort (Tissue cohort, n = 19) of endometriosis patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Paired-endometrial and -endometriotic biopsies were obtained from women with endometriosis (ASRM stage III-IV) undergoing laparoscopic surgery. Control endometria were obtained from healthy volunteers. Isolated CD73+CD90+CD105+ SC were evaluated for the presence of known endometrial surface markers, colony forming efficiency, multi-lineage differentiation, cell cycle distribution and 3D-spheroid formation capacity. Targeted RT-PCR arrays, along with hierarchical and multivariate clustering tools, were used to determine both intergroup and intragroup gene expression variability for stem cell and cancer-associated markers, in both SC+ and tissue cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: Isolated and expanded SC+ from both control and patient groups showed significantly higher surface expression of W5C5+, clonal expansion and 3D-spheroid formation capacity (P < 0.05) compared with SC-. The SC+ cells also undergo mesenchymal lineage differentiation, unlike SC-. Gene expression from paired-endometriosis samples showed significant downregulation of PTEN, ARID1A and TNFα (P < 0.05) in endometrioma compared with paired-endometrium SC+ samples. Hierarchical and multivariate clustering from both SC+ and tissue cohorts together identified 4 out of 30 endometrioma samples with aberrant expression of stem cell and cancer-associated genes, such as KIT, HIF2α and E-cadherin, altered expression ratio of ER-ß/ER-α and downregulation of tumour suppressor genes (PTEN and ARID1A). Thus, we speculate that above changes may be potentially relevant to the development of EAOC. LARGE-SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: As the reported frequency of EAOC is very low, we did not have access to those samples in our study. Moreover, by adopting a targeted gene array approach, we might have missed several other potentially-relevant genes associated with EAOC pathogenesis. The above panel of markers should be further validated in archived tissue samples from women with endometriosis who later in life developed EAOC. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge gained from this study, with further confirmation on EAOC cases, may help in developing screening methods to identify women with increased risk of EAOC. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Swedish Research Council (2012-2844), a joint grant from Stockholm County and Karolinska Institutet (ALF), RGD network at Karolinska Institutet, Karolinska Institutet for doctoral education (KID), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695), Horizon 2020 innovation program (WIDENLIFE, 692065), European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways funding (IAPP, SARM, EU324509) and MSCA-RISE-2015 project MOMENDO (691058). All authors have no competing interest.

Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form.

The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity.

GSTM1 gene expression correlates to leiomyoma volume regression in response to mifepristone treatment.

UNLABELLED: Progesterone receptor modulators, such as mifepristone are useful and well tolerated in reducing leiomyoma volume although with large individual variation. The objective of this study was to investigate the molecular basis for the observed leiomyoma volume reduction, in response to mifepristone treatment and explore a possible molecular marker for the selective usage of mifepristone in leiomyoma patients. Premenopausal women (N = 14) were treated with mifepristone 50 mg, every other day for 12 weeks prior to surgery. Women were arbitrarily sub-grouped as good (N = 4), poor (N = 4) responders to treatment or intermediate respondents (N = 3). Total RNA was extracted from leiomyoma tissue, after surgical removal of the tumour and the differential expression of genes were analysed by microarray. The results were analysed using Ingenuity Pathway Analysis software. The glutathione pathway was the most significantly altered canonical pathway in which the glutathione-s transferase mu 1 (GSTM1) gene was significantly over expressed (+8.03 folds) among the good responders compared to non responders. This was further confirmed by Real time PCR (p = 0.024). Correlation of immunoreactive scores (IRS) for GSTM1 accumulation in leiomyoma tissue was seen with base line volume change of leiomyoma R = -0.8 (p = 0.011). Furthermore the accumulation of protein GSTM1 analysed by Western Blot correlated significantly with the percentual leiomyoma volume change R = -0.82 (p = 0.004). Deletion of the GSTM1 gene in leiomyoma biopsies was found in 50% of the mifepristone treated cases, with lower presence of the GSTM1 protein. The findings support a significant role for GSTM1 in leiomyoma volume reduction induced by mifepristone and explain the observed individual variation in this response. Furthermore the finding could be useful to further explore GSTM1 as a biomarker for tailoring medical treatment of uterine leiomyomas for optimizing the response to treatment. CLINICAL TRIALS IDENTIFIER: www.clinicaltrials.gov: NCT00579475, Protocol date: November 2004. http://clinicaltrials.gov/ct2/show/NCT00579475.

Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner.

OBJECTIVE: To study the role of a synthetic insulin-like growth factor-I receptor (IGF-IR) antagonist, picropodophyllin, for mouse preimplantation embryo development in vivo and in vitro. DESIGN: In vitro and in vivo study. SETTING: Hospital-based research unit. ANIMALS: FVB/N mice and mouse embryos. INTERVENTION(S): The effect of picropodophyllin in mouse embryo development in vivo and in vitro, immunohistochemistry, ELISA, polymerase chain reaction. MAIN OUTCOME MEASURE(S): Embryo development, presence of IGF-IR, messenger RNA expression, IGF-I synthesis. RESULT(S): The effect of picropodophyllin on embryo development in vitro and in vivo was not reversible. Mice treated with picropodophyllin 1 to 3 days after mating had a reduced number of blastocysts, 40.5% versus 78.8%, and a higher number of embryos with delayed development, 48.6% versus 11.5%. Insulin-like growth factor-IR protein is present in both phosphorylated and nonphosphorylated form at all stages of embryo development. The relative IGF-IR messenger RNA expression was highest in the oocyte and reduced during development to blastocyst stage. Insulin-like growth factor-I in culture media was reduced after picropodophyllin treatment. CONCLUSION(S): We conclude that IGF-I has an important role in normal mouse embryo development and that its receptor plays an essential role in the embryonic genome activation process.

Expressions of steroid receptors and Ki67 in first-trimester decidua and chorionic villi exposed to levonorgestrel used for emergency contraception.

Levonorgestrel (1.5 mg) is commonly used for emergency contraception to prevent an unwanted pregnancy after an unprotected intercourse. We found that postovulatory administration of 1.5 mg of levonorgestrel to women with a subsequent or existing early pregnancy did not affect the immunohistochemical expressions of estrogen receptors (ER(alpha), ER(beta)), P receptors (PR(B), PR(A+B)), androgen receptor (AR), or proliferation index Ki67 in the first-trimester decidua and chorionic villi.