Implantation Failure In Women Research Articles

Dysregulated leukemia inhibitory factor and its receptor regulated signal transducers and activators of transcription 3 pathway: a possible cause for repeated implantation failure in women with dormant genital tuberculosis?

To investigate the influence of dormant Mycobacterium tuberculosis on the expression of various endometrial receptivity markers and leukemia inhibitory factor (LIF)-signal transducers and activators of transcription 3 (STAT3) signaling pathway. Expression of endometrial receptivity markers and LIF-STAT3 signaling in invitro decidualized human endometrial stromal cells (hESC) treated with 65kDa mycobacterial heat shock protein (HSP65) is also explored. A prospective study. Tertiary care hospital and reproductive health research unit. Endometrial tissue samples were collected from 38 women who tested positive for Mycobacterium tuberculosis and 30 normal women with proven fertility undergoing sterilization. Invitro decidualization of hESC was performed. Endometrial biopsies collected from all women during implantation window and treatment of hESC with HSP65. Measurement of various endometrial receptivity markers including αvβ3 integrin, E-cadherin, MECA-79, mucin-1, and pinopodes and LIF/LIFR-STAT3 signaling molecules expressed in the endometrium of women with dormant genital tuberculosis (GTB) during implantation window and measured also in HSP65-treated hESC. Significantly reduced levels of endometrial receptivity markers LIF, LIFR, and pSTAT3 were observed in endometrium of women with dormant GTB as compared with controls. A similar trend was observed under invitro conditions with decreased level of phosphorylated STAT3 in HSP65-treated hESC. However, no change in the expression of endometrial receptivity markers under invitro conditions was observed. Our findings suggest that endometrium of women with dormant GTB is associated with poor receptivity, as evidenced by reduced receptivity markers and aberrant LIF-STAT3 signaling. Invitro treatment of hESC with HSP65 also confirms compromised endometrial decidualization.

Recurrent ectopic pregnancy history is one of the risk factors of intrauterine implantation failure in women undergoing frozen-thawed embryo transfer cycles

Recurrent ectopic pregnancy history is one of the risk factors of intrauterine implantation failure in women undergoing frozen-thawed embryo transfer cycles

Urinary concentrations of bisphenol a and their association with indicators of implantation in IVF couples

Bisphenol A (BPA) is a synthetic chemical widely used in the production of polycarbonate plastics and epoxy resins found in numerous consumer products and lead to human exposure. There was a report suggesting positive linear dose-response association between BPA urinary concentrations and implantation failure in women undergoing in vitro fertilization (IVF) (1 , 2 , 3). We evaluated the association of female and male urinary BPA concentrations with various indicators of IVF.

The vascular endothelial growth factor (VEGF) +405 G/C polymorphism and its relationship with recurrent implantation failure in women in an IVF programme with ICSI

Successful embryo implantation depends on trophoblast proliferation, migration and, lastly, invasion of the endometrium (to anchor the trophoblast to the uterus). This invasion is mediated by locally produced soluble factors. Of these, vascular endothelial growth factor (VEGF) is the best characterized regulator of angiogenesis. Here, we investigate the association between the VEGF + 405 C/G genotype and the recurrence of embryo implantation failure in women undergoing in vitro fertilization (IVF) program with intracytoplasmic sperm injection (ICSI). Forty women with recurrent implantation failure defined by absence of pregnancy after transfer of more than 10 embryos and 131 women control, with at least one live birth after the transfer of fewer than 10 embryos were included. Genomic DNA was analysed with an allele-specific polymerase chain reaction and a Chi-2 test was used to compare the respective VEGF + 405 C/G genotype frequencies in cases and controls. The frequency of the VEGF +405C/C genotype was higher in women with recurrent implantation failure after ICSI-embryo transfer than in controls (17.5% and 5.3%, respectively, p = 0.01). The VEGF +405G/C polymorphism may influence embryo implantation and VEGF + 405 C/C genotype may predispose to recurrent implantation failure after ICSI-ET.

Urinary bisphenol A and implantation failure among women undergoing in vitro fertilization

Bisphenol A (BPA) is a synthetic chemical used in the production of polycarbonate plastic and epoxy resins found in consumer products. In experimental animals, BPA has been associated with embryo implantation failure and reduced litter size. We evaluated the association of urinary BPA concentrations with risk of implantation failure in women undergoing in vitro fertilization (IVF).

The use of G-CSF for implantation failure in IVF: a clinical trial

We previously showed that G-CSF may be useful in the treatment of recurrent abortion. The G-CSF is a cytokyne promoting leukocyte growth, but also troflobast development. In this sudy we tested this cytokyne as a treatment for recurrent implantation failure in women undergoing IVF. A randomized controlled clinical trial The study was conducted to the CERM, Rome, Italy, from the January 2008 to December 2010 on 89 women with recurrent implantation failure after IVF cycles. The study was approved by IRB. The inclusion criteria were, at least three previous failed IVF attempts where at least 7 good embryos were transferred, women less than 39 years old, absence of systemic diseases. These women underwent IVF cycle and they were randomly divided in two groups: one (45 women) treated with subcutaneous G-CSF 1.5mg/Kg /daily (60-100mg) from the day of transfer to the day of β-hCG test, and if it was positive the treatment was continued for other 40 days; the group of control was treated with subcutaneous saline solution infusion in the same way of the study group. Primary outcomes were considered: pregnancy rate and the of β-hCG levels at 14/21/28 and 35 days after embryo transfer. Statistical analysis was performed using the Mann Whitney U test for continuous variables and Fisher exact test for comparison of proportions. The epidemiological data of patient groups did not show statistically significant differences. The pregnancy rate in the group treated with G-CSF was of 42.2% (16/45) whereas in the control group 15.9% (7/44), this difference was statistically significant P=0.0176. The levels of β-hCG after 14 days, 21 days 28 days and 35 days from embryo transfer were statistically significant higher in the pregnancies obtained in the group treated with G-CSF than in control group pregnancies (P<0.001). No severe side effects of treatment were observed. The clinical use of G-CSF in women experienced implantation failure may be useful, but more studies are needed.

P.26 Evaluation of risk factors in women with breast carcinoma who developed venous thromboembolism

P.26 Evaluation of risk factors in women with breast carcinoma who developed venous thromboembolism

P.28 Factor V Leiden, PT-G20210A, MTHFR C677T mutations and the risk of implantation failure in women undergoing assisted reproductive procedures (IVF or ICSI)

P.28 Factor V Leiden, PT-G20210A, MTHFR C677T mutations and the risk of implantation failure in women undergoing assisted reproductive procedures (IVF or ICSI)

Urinary metabolites of Di(2-ethylhexyl) phthalate are associated with increased risk of implantation failure among women undergoing IVF

OBJECTIVE: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in consumer products. In rodent studies, MEHP (a metabolite of DEHP) was associated with implantation failure and reduced litter size. We investigated the association between urinary DEHP metabolites and implantation failure in women undergoing IVF.

Activin A regulates trophoblast cell adhesive properties: implications for implantation failure in women with endometriosis-associated infertility

During implantation, the embryo adheres to the endometrium via cell adhesion molecules (CAMs) present on blastocyst trophectoderm and endometrial epithelial cells. CAMs, including integrins and extracellular matrix (ECM) ligands, are most likely regulated by hormones, cytokines and growth factors. We hypothesized first that activin A affects the adhesive properties of trophoblast cells and second that alterations in dimeric activin A levels in the uterine cavity could disrupt adhesion, thereby causing implantation failure. This study examined effects of activin A on trophoblast cell adhesion and measured activin A levels in secretory phase uterine washings from women with and without endometriosis (EOS). Activin receptor expression on trophoblast (HTR8) cells was examined by RT-PCR, and adhesive molecules measured by integrin antibody and cell-matrix adhesion assays. Dimeric activin A was measured (enzyme-linked immunosorbent assay) in uterine washings (14 controls and 23 EOS), and betaA-subunit localization was verified in endometrial tissues. Activin receptors are expressed by HTR8 cells. Activin A activated Smad2 in a concentration-dependent manner which was blocked by an activin receptor inhibitor (SB431542). Following activin A treatment (50 ng/ml for 24 h), trophoblast cell surface integrins alpha1 alpha2 alpha3 alpha5, beta1, beta2, beta4 and alphavbeta5 were decreased, as was cell binding to the ECM ligands, fibronectin, collagen IV and collagen I (P < 0.05). Activin A was detected in 56.5% of EOS and 21.4% of control washings, with measured levels from 42 to 8481 pg/ml (not significantly different). Decreased trophoblast CAM production and adhesion could be caused by dysregulated local activin A levels and may contribute to implantation failure. This could explain, in part, the infertility observed in women with EOS.

Endometrial Collagen Fibril Hyperplasia is Associated with Implantation Failure in Women Undergoing IVF-ET

Objective To analyse the effects of controlled ovarian hyperstimulation (COH) on the endometrial expression of collagen fibril (CF) during the peri-implantation period in patients undergoing IVF, and its relation to endometrial receptivity (ER) in repeated implantation failure(RIF). Methods Peripheral blood and endometrial biopsies were obtained from 45 infertile women on days 5, 7 or 9 after oocytes retrieval or ovulation in a stimulated cycle (SC) and natural cycle (NC) respectively. CF was assayed by transmission electron microscope and quantified by modified Masson dyeing. The outcome of subsequent embryo transfer(ET) was observed. Results Levels of both E 2 and progesterone were higher in the peripheral blood in SC than in NC. Also the expression of CF in the stroma in each secretory phase was increased significantly in SC (P Conclusion Imbalance of production and degradation of endometrial CF in the secretory phase resulting from COH may be the cause of defective ER and implantation failure in some RIF patients. Endometrial curettage may improve implantation rate by inducing appropriate CF hyperplasia and degradation.

529. PROPROTEIN CONVERTASE 6 PLAYS A CRITICAL ROLE IN MODULATING THE HUMAN ENDOMETRIAL EPITHELIUM FOR RECEPTIVITY AND IMPLANTATION

Successful embryo implantation is an important step in establishing pregnancy, requiring a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, but the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation, in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical “master switch” molecules. The present study aimed to determine whether PC6 is a critical regulator in the endometrial epithelium for receptivity and implantation. We examined whether endometrial epithelial PC6 dys-regulation is associated with implantation failure in women and whether knockdown of PC6 by siRNA in human endometrial epithelial cells affects embryo adhesion in a cell culture model. Endometrial PC6 expression was assessed by immunohistochemistry in the mid-secretory phase of the menstrual cycle (receptive phase) in two unique clinical cohorts comprising women of known fertility and infertility (with no obvious gynecological disorders, and with fertile males). Endometrial epithelial PC6 levels were significantly lower in infertile vs fertile women in both cohorts. To further establish that PC6 is important for receptivity, a cell model relevant to human implantation was used involving co-culture of uterine epithelial cells with mouse embryos. The epithelial cells were stably transfected with PC6 siRNA and PC6 knock down was confirmed at the levels of mRNA, protein, and activity by real-time RT-PCR, Western blotting and activity assay respectively. Embryos readily adhered to normal epithelial cells, but the adhesion was significantly reduced in the PC6 knockdown epithelial cells. We are currently using proteomics technology to identify the pathways affected by PC6 knockdown. These results strongly suggest that PC6 plays a critical role in modulating the human endometrial epithelium for receptivity and implantation.

Placental Thrombosis in Experimental Anticardiolipin Antibodies‐Mediated Intrauterine Fetal Death

Anticardiolipin (aCL) antibodies are associated with stillbirths, recurrent miscarriages and recurrent in vitro fertilization implantation failure in women. Previous animal studies have demonstrated that these antibodies can cause early fetal demise and implantation failure in mice, but most previous studies have not allowed the immunized mice to proceed to the full term of gestation. Mice were immunized with either cardiolipin alone or cardiolipin in combination with beta2-glycoprotein I (beta2GPI) and have studied the effects of these antibodies on pregnancies which were allowed to progress to term. Immunization with cardiolipin alone induced significant levels of anticardiolipin antibodies in mice, but immunization with a combination of cardiolipin and beta2GPI produced even higher levels of antibodies. Mice with elevated levels of anticardiolipin antibodies had poor pregnancy outcomes. This study confirms previous results that anticardiolipin antibodies cause early pregnancy losses and also demonstrates that these antibodies cause stillbirth-like late fetal demise. This study further demonstrated that very high levels of anticardiolipin antibodies cause intrauterine death by facilitating the thrombotic episode in placenta. The present study concludes that the possible mechanism involves in stillbirth of aCL is possibly because of the thrombotic events of placenta.

Differentially expressed genes implicated in unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion occurs in approximately 3% of women with diagnosed pregnancies. The etiology in approximately 40% of recurrent spontaneous abortion is unexplained. To elucidate unexplained recurrent spontaneous abortion at the molecular level, we systemically identified differentially expressed genes during implantation window period in unexplained recurrent spontaneous abortion and characterized their functions in a human endometrial cell line. Expression levels of implantation-related genes selected from previously reported, various microarray data were determined to identify differentially expressed genes between normal fertile and unexplained recurrent spontaneous abortion subjects by real-time quantitative RT-PCR. Of 29 implantation-related genes, the transcript levels of cellular retinoic acid binding protein 2 and olfactomedin 1 were higher, whereas that of complement component 4 binding protein alpha was lower in subjects with unexplained recurrent spontaneous abortion, compared to normal fertile subjects. A correlation was evident between the transcript and protein levels of complement component 4 binding protein alpha and cellular retinoic acid binding protein 2. Expression of cellular retinoic acid binding protein 2 was positively correlated with retinoic acid-related genes in normal fertile subjects, but no significant association was observed in unexplained recurrent spontaneous abortion subjects. In relation to complement component 4 binding protein alpha, C5a receptor protein level was significantly higher in subjects with unexplained recurrent spontaneous abortion. Stable expression of cellular retinoic acid binding protein 2 and olfactomedin 1 in a human endometrial cell line inhibited cell growth and induced cell accumulation in the S and G 2-M phase fractions, but did not trigger apoptosis. This study represents the first systematic identification of differentially expressed genes in unexplained recurrent spontaneous abortion. Defective cell growth by the differentially expressed genes suggests their implication in implantation failure in women with unexplained recurrent spontaneous abortion.

Deficient expression of monoamine oxidase A in the endometrium is associated with implantation failure in women participating as recipients in oocyte donation

Successful implantation depends both on the quality of the embryo and on the endometrial receptivity. The latter depends on progesterone-induced changes in gene expression, a process that has been characterized by microarray analysis. One of the genes whose transcription appears to be enhanced during the receptive period is monoamine oxidase A (MAO-A). Our first objective was to confirm the increased expression of MAO-A in the endometrium during the receptive phase of spontaneous normal cycles using real time PCR and immunofluorescence. The second objective was to examine the endometrial expression of MAO-A during the receptive phase induced by exogenous estradiol (E(2)) and progesterone in patients whose endometrium was shown to have been either receptive or non-receptive to embryo implantation in repeated cycles of oocyte donation. Results showed that MAO-A transcript levels increased between the pre-receptive (LH+3) and receptive phase (LH+7) in all spontaneous cycles examined, with a median increase of 25-fold. Immunofluorescent labelling demonstrated MAO-A localization to the glandular and luminal epithelium with an increasing positive score between LH+3 and LH+7. Conversely, prior failure of embryo implantation was associated with a 29-fold decrease in MAO-A mRNA levels and a substantial reduction in MAO-A protein immunofluorescent label score. These results show a strong association between endometrial receptivity and MAO-A expression in the endometrial epithelium, suggesting an important role for this enzyme in normal implantation.

Human progesterone receptor polymorphisms and implantation failure during in vitro fertilization

Objective Polymorphic variants of the human progesterone receptor gene have been described, but their potential influence on in vitro fertilization outcome have not been studied. Study design DNA was available from 317 women (cases) who had ≥2 embryo transfers without a clinical pregnancy and from 288 women (control subjects) who became pregnant after 1 in vitro fertilization attempt. With protocols that were specific for H770H (C/T genotype) and +331G/A polymorphism, amplification of polymorphism fragments, digestion with restriction enzymes, and gel visualization were performed. Results Allele frequencies for the 2 variants were 15.5% for H770H T and 4.1% for +331A in cases and 17.9% and 3.6% in control subjects, respectively. Both polymorphisms were rare among non-white subjects. Possession of a H770H T allele was associated with a decreased risk for implantation failure, which was nonsignificant overall but significant in women aged <35 years (adjusted relative risk, 0.52 [95% CI, 0.28, 0.97]). Possession of a +331A allele was associated with an increased risk of implantation failure, which was nonsignificant overall but significant in women who weighed <135 pounds (adjusted relative risk, 3.85 [95% CI, 1.20, 12.30]). The +331G/A polymorphism tended to increase the risk for implantation failure in women with an H770H C/C genotype, but not in women with a C/T or TT genotype ( P = .09). Among white women who were H770H C/C, there was a significant trend ( P = .03) in the proportion of +331G/A carriers as the number of implantation failures increased, from 8.6% of women with 0 failed attempts to 40% among women with ≥5 failed attempts. Conclusion Although human progesterone receptor polymorphisms do not clearly affect the risk for implantation failure in most women who undergo in vitro fertilization, the likelihood that a woman carries the +331G/A human progesterone receptor polymorphism increases with the number of failed attempts at implantation.

Antinuclear antibody reduces the pregnancy rate in the first IVF-ET treatment cycle but not the cumulative pregnancy rate without specific medication.

It has been shown that the presence of antinuclear antibody (ANA) might reduce pregnancy rates after in vitro fertilization-embryo transfer (IVF-ET). However, the mechanism of implantation failure by ANA has not yet been clarified. This study was performed to investigate the impact of ANA on pregnancy rates after IVF-ET, and the necessity of specific medication for infertile women who have ANA in their sera. A total of 108 infertile women were treated by IVF-ET or intracytoplasmic sperm injection (ICSI)-ET. ANA was examined by an indirect fluorescent antibody procedure. Data from women under 40 years old were analyzed retrospectively. The implantation rates per embryo transferred in the first treatment cycles were 14.8% (eight of 54) and 32.4% (33 of 102), in women with and without ANA, respectively. There was a significant difference in the implantation rates between the two groups (P < 0.05). The pregnancy rates per ET in the first treatment cycles were 28% (seven of 25) and 54.2% (26 of 48), respectively. There was also a significant difference in the pregnancy rates between the two groups (P < 0.05). Afterwards, treatments with IVF-ET or ICSI-ET were repeatedly performed for unsuccessful patients, without any specific medication for ANA. The average ET cycles were 1.80 +/- 1.13 and 1.27 +/- 0.54, in women with and without ANA, respectively. The cumulative pregnancy rates per patient were 68% (17 of 25) and 55.6% (35 of 63), respectively. There was no significant difference in the overall pregnancy rates between the two groups. These findings suggest that ANA might have an impact on implantation failure in women treated by IVF-ET or ICSI-ET. ANA reduced the pregnancy rates in the first IVF-ET or ICSI-ET cycles but not the cumulative pregnancy rates without medication. This indicates that the mechanisms of implantation failure by ANA could be solved, and effective and safe medication should be developed for better implantation rates, especially in the first treatment cycle.

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

IMMUNOLOGY OF NORMAL PREGNANCY

Reproductive immunology has become a mature discipline. It had its birth in the field of transplantation immunology. Scientists assumed that the fetus was a transplant and would follow the laws of rejection or acceptance, but many of these theories and hypotheses were difficult to validate and had to be discarded. Today, sophisticated immunological methods have been used by scientists to define more clearly the mechanisms of immunoregulation during a normal pregnancy, a failing pregnancy, and during implantation failure in women with idiopathic infertility undergoing in vitro fertilization. It is becoming clear that the embryonic cell, which attaches the embryo to the uterine lining and eventually forms the placenta, initiates responses that stimulate a T helper 2 (Th-2) suppressive response (Fig. 1). Human leukocyte antigen (HLA) DQ A1 compatibility between the mother and the fetus results in the destruction of the embryo during the early stages of pregnancy.101 In this circumstance, compatibility seems to breed autoimmune cellular and humoral immune contempt that becomes more aggressive the greater the number of pregnancy failures the woman experiences. Figures 1 and 2 outline in simple form how the trophoblast chooses the immune responses that protect versus those that are cytotoxic and result in failure.