Low dose prednisolone administration in routine ICSI patients does not improve pregnancy and implantation rates

BACKGROUND: Glucocorticoids have been used in conjunction with zona dissection to improve pregnancy and implantation rates in IVF patients. The aim of this prospective randomized study was to evaluate the effect of low-dose prednisolone in addition to the standard protocol, on pregnancy and implantation rates in routine ICSI patients before and after embryo replacement. METHODS: A total of 313 patients in 360 consecutive cycles (patients <39 years old and with three or less than three ICSI attempts) performed at our centre were randomly assigned by computer-generated list to receive either prednisolone (10 mg/day in two divided doses), starting on the first day of ovarian stimulation and continuing for 4 weeks (group A), or no treatment (group B). RESULTS: The mean age, number of previously failed IVF attempts, basal FSH levels and the mean rank of trials were comparable between groups A and B. The mean (+/- SD) number of metaphase II oocytes retrieved (11.9 +/- 5.5 versus 12.0 +/- 5.1), 2-pronuclei fertilization rate (67.2 versus 65.8%), the pregnancy and the implantation rates were not different between the study and control groups (49.0 and 23.6% versus 50.0 and 23.3% respectively). CONCLUSION: Low-dose prednisolone treatment in addition to the standard protocol before and after embryo replacement does not appear to have a significant effect on pregnancy or implantation rates.     

The role of fibronectin in tumor implantation at surgical sites

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 µg/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.     

Embryo transfer under ultrasound guidance improves pregnancy rates after in-vitro fertilization

Between October 1998 and January 1999, we examined the influence of ultrasound guidance in embryo transfer on pregnancy rate in 362 patients from our in-vitro fertilization (IVF)-embryo transfer programme. These patients were prospectively randomized into two groups: 182 had ultrasound-guided embryo replacement, and 180 had clinical touch embryo transfer. There were no statistically significant differences between the two groups with respect to age, cause of infertility and in the characteristics of the IVF cycle. The pregnancy rate was significantly higher among the ultrasound-guided embryo transfer group (50%) compared with the clinical touch group (33.7%) (P < 0.002). Furthermore, there was also a significant increase in the implantation rate: 25.3% in the ultrasound group compared with 18.1% in the clinical touch group (P < 0.05). In conclusion, ultrasound assistance in embryo transfer significantly improved pregnancy and implantation rates in IVF.     

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.     

Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated

Partial zona dissection (PZD) of human oocytes facilitates spermpenetration through mechanically made holes in the zona pellucida.Only 1 of 69 eggs was damaged when sucrose was used to shrinkthe ooplasm during micromanipulation. The fertilization rateof micromanipulated oocytes in 18 couples with male factor infertilitywas 68% (34/50), which compared favourably with inseminationof non-micromanipulated controls (21/45, 47%). PZD was advantageousin oligozoospermic patients, but not in cases of asthenozoospermia,combined semen problems or immunological infertility. Threetwin and two singleton pregnancies resulted following replacementof 23 micromanipulated and eight control embryos in 14 patients.No differences in embryo morphology and development rates werefound between the micromanipulated and control groups. The incidenceof polyspermy in couples with abnormal semen analyses was relativelylow (<20%) possibly due to partial activation of the oocytesfollowing exposure to sucrose. Polyspermy was high (57%) innormozoospermic patients with either immunological infertility(n= 3) or failure of fertilization in previous cycles (n= 4).In the three immunological patients, nine of 11 hyaluronidaseand sucrose-exposed control embryos fertilized and six implanted,possibly indicating that cumulus and corona cells are contributingfactors inhibiting fertilization in such cases.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Removal of hydrosalpinges increases endometrial leukaemia inhibitory factor (LIF) expression at the time of the implantation window

BACKGROUND: The presence of hydrosalpinges is associated with lower implantation and pregnancy rates in women undergoing IVF-embryo transfer, while salpingectomy improves these parameters. Although the mechanism by which hydrosalpinges affects fertility is not entirely understood, an adverse effect on endometrial receptivity has been postulated. In this study, we hypothesized that the adverse effects of hydrosalpinges on fertility may be in part mediated by inappropriate endometrial expression of leukaemia inhibitory factor (LIF), a cytokine implicated in implantation. METHODS: In order to test our hypothesis, we prospectively examined the expression of LIF during the window of implantation in the endometrium of infertile women (n = 10) with hydrosalpinges prior to and following salpingectomy and of fertile controls (n = 10) by Western blotting and immunohistochemistry. RESULTS: LIF expression was significantly lower in infertile women with hydrosalpinges compared with fertile controls (P < 0.05). Salpingectomy resulted in an increase in LIF expression in eight out of 10 women with hydrosalpinges. LIF levels were increased by 231 +/-49% (mean +/- SEM) following salpingectomy. Immunohistochemical analysis confirmed the Western blot findings. The increased LIF immunoreactivity was predominantly localized to luminal and glandular epithelial cells. CONCLUSIONS: Our findings suggest that observed benefit from salpingectomy in infertile women with hydrosalpinges may be in part mediated by the up-regulation of endometrial LIF expression.     

Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus

Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by approximately 2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This "FAAH activator" was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A(2), C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B(4) or prostaglandins E(2) and F(2)alpha had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated.     

Implantation, interception and contraception

The factors involved in post-fertilization events leading toimplantation in mammals are discussed with special referenceto potential forms of interception. The stages of embryonicgrowth until implantation are considered initially. The growthand differentiation of the uterine endometrium is then described,followed by the events occurring during the apposition and invasionof the implanting embryo. Several potential approaches to newforms of interception are considered, and the advantages anddisadvantages of each of them are evaluated. Among them, newvaccines against the zona pellucida, inactivation of the secretionsof the blastocyst, hatching, the activity of the pinopodes,and the endometrial proteins produced in the secretory phaseseem to offer various and varied targets. Some existing methodsof fertility regulation may act by affecting these stages ofdevelopment, e.g. RU486 may interfere with pinopod function.Various physiological and embryonic consequences of interferingwith these stages of pregnancy are discussed.     

Differentiation of endometrial stromal cells in vitro: down-regulation of suppression of the cell cycle inhibitor p57 by HOXA10?

Decidualization is a critical step during embryo implantation that is characterized by the differentiation of endometrial stromal cells (ESC) into decidua cells. However, the mechanism of differentiation remains largely unknown. Previously, it has been shown that the null function of homeo box A10 (HOXA10) causes defects in both implantation and decidualization, suggesting that the HOXA10 signalling pathway is likely to be involved in uterine decidualization. In the present study, we determined the expression and subcellular distribution of HOXA10 and its downstream molecule, p57, in ESC during in vitro decidualization induced by a combination of 8-bromo-cAMP and medroxyprogesterone acetate. We demonstrated that the HOXA10 was down-regulated while in contrast, p57 was up-regulated in the process of decidualization. Immunocytochemistry and transient expression of the HOXA10 tagged with green fluorescence protein revealed that there were no differences in the HOXA10 subcellular localization between the induced and non-induced ESC. Our results suggest that the down-regulation of HOXA10 may contribute to increased p57 and that up-regulation of p57 likely plays an important role in ESC differentiation in the process of decidualization. The progesterone receptor pathway may participate in promoting ESC to exit the cell cycle and enter differentiation.