J.S. Kruessel

Effect of oral N‐acetyl cysteine on recurrent preterm labor following treatment for bacterial vaginosis

To evaluate the effect of N‐acetyl cysteine (NAC) on gestational age at delivery in women with previous preterm labor and bacterial vaginosis.

Expression of the vascular endothelial growth factor receptor neuropilin-1 in the human endometrium

Angiogenesis is a key process in the endometrium which undergoes dramatic changes during the menstrual cycle. Molecules such as vascular endothelial growth factor (VEGF), acting via two tyrosine kinase family receptors (VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1]), are potent modulators of angiogenesis and vascular remodelling in the endometrium. Recently, neuropilin-1 (NRP-1) was shown to be expressed in endothelial cells binding VEGF(165) and therewith enhancing the binding of VEGF(165) to VEGFR2. This suggests that NRP-1, in addition to the known VEGF receptors, may play an important role in VEGF-induced angiogenesis. In this study, the expression of NRP-1 in the cycling human endometrium has been investigated by reverse transcription (RT)-polymerase chain reaction (RT-PCR), semi-quantitative competitive RT-PCR (RT-cPCR) and immunohistochemical staining. NRP-1 was expressed in all 32 endometrium samples throughout the menstrual cycle. However, samples from the proliferative phase showed significantly higher expression levels of NRP-1 mRNA compared to samples from the secretory phase (t/c-ratio 2.13 vs. 0.84, p<0.05). Immunohistochemistry confirmed the results showing increased NRP-1 staining in vascular endothelium, glandular epithelium and stromal cells of the proliferative phase endometrium. This study demonstrates mRNA and protein expression of NRP-1 in human endometrium samples throughout the menstrual cycle. The enhanced expression of NRP-1 in the proliferative phase suggests that it may participate in hormonally regulated changes of endometrial angiogenesis, preparing the endometrium for the implantation of an embryo. NRP-1 expression might act as a co-factor for VEGF(165) enhancing the angiogenic stimulus.

Interleukin-1 system in the human fallopian tube-No spatial but a temporal regulation of mRNA and protein expression.

The human fallopian tube provides the environment for the first 5 days of embryonic development in vivo. The IL-1 system is involved in human embryo implantation. This study aimed to investigate IL-1beta, IL-1ra and IL-1R tI expression within the length of the human fallopian tube on mRNA- and protein-level in samples from proliferative versus secretory phase, postmenopause (PMP) samples and samples from intra- (IUP) and extrauterine pregnancies (EUP) to examine possible spatial and hormonal induced changes (fimbrial, ampullary and isthmic tube segments). On mRNA-level, IL-1beta was expressed in all samples except in PMP. IL-1R tI could be detected in all samples whereas IL-1ra was only expressed in secretory phase and the IUP sample. Immunohistochemically we could detect IL-1beta and IL-1R t1 protein in all proliferative and secretory phase samples with maximum intensity in secretory phase samples whereas IL-1ra was expressed in secretory phase samples only. Overall no spatial but temporal differences possibly due to hormonal changes could be observed suggesting a precise regulation of the IL-1 system, especially for IL-1ra and moreover a stable molecular architecture within the full length of the fallopian tube.

Decidualization and Syndecan-1 Knock Down Sensitize Endometrial Stromal Cells to Apoptosis Induced by Embryonic Stimuli

Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1), and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the light of an already described influence on pregnancy disorders and therefore might provide a useful clinical tool in the future to prevent pregnancy complications provoked by inadequate implantation.

Syndecan-1 knockdown in endometrial epithelial cells alters their apoptotic protein profile and enhances the inducibility of apoptosis

Endometrial epithelial cells are known to undergo apoptosis during trophoblast invasion. We postulate that the cell surface molecule Syndecan-1 which is expressed on endometrial cells and syncytiotrophoblast is important for implantation in general and especially for induction of maternal cell apoptosis during trophoblast invasion because Syndecan-1s influence on apoptotic susceptibility of cancer cells is already described in the literature. Using the human endometrial epithelial cell line RL95-2, a new stable cell line with Syndecan-1 knockdown was generated. Via antibody array analysis, a significant decrease in the expression of anti-apoptotic proteins like inhibitors of apoptosis, Clusterin, heme oxygenase (HO-2), heat shock protein (HSP)27 and -70 and Survivin due to the Syndecan-1 knockdown was discovered. Correspondingly, active Caspase-3 as an indicator for apoptosis was increased more severely in these cells compared with unmodified RL95-2 after treatment with implantation-related stimuli, which are the cytokines interleukin-1β, interferon-γ, tumor necrosis factor-α and transforming growth factor-β1 and an anti-Fas antibody. Furthermore, a treatment with a combination of all factors caused a higher Caspase-3 induction compared with each single treatment. These results demonstrate that Syndecan-1 is involved in the control of apoptosis in RL95-2 cells and therefore may affect the fine tuning of apoptosis in endometrial epithelium regulating the embryos invasion depth as a crucial step for regular implantation followed by successful pregnancy.

In vitro culture does not alter the expression of vascular endothelial growth factor and its receptors in single murine preimplantation embryos.

Background: In vitro culture of embryos, as widely used in assisted reproduction techniques, may influence embryonic development and subsequently the establishment of pregnancy. The aim of this study was to determine a potential influence of the in vitroculture regarding VEGF, VEGFR1 and VEGFR2 mRNA expression in developing single mouse embryos. Methods: Murine embryos were isolated on day 1 post coitus (p.c.) and cultivated for a developmental time course followed by examination for mRNA expression using RT-nested PCR. Furthermore, in vitro cultured blastocysts were compared to in vivo development at 101 h p.c. Results: At 101 h p.c. there were no significant differences between in vivo and in vitro cultured blastocysts regarding the expression of VEGF and its receptors. In the developmental time course, VEGF expression increased up to 94% in late blastocysts whereas the VEGF receptor expression remained low. Conclusions: This study showed that the in vitro culture did not alter the embryonic VEGF and VEGFR mRNA expression reassuring that the culture conditions in assisted reproduction techniques are well suited for maintaining the VEGF mRNA expression profile. Additionally, nearly 100% VEGF expression in late blastocysts highlights its importance for angiogenesis induction at the fetal-maternal interface.