Long-Term Effects of ART on the Health of the Offspring.

Assisted reproductive technologies (ART) significantly increase the chance of successful pregnancy and live birth in infertile couples. The different procedures for ART, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI), and gamete intrafallopian tube transfer (GIFT), are widely used to overcome infertility-related problems. In spite of its inarguable usefulness, concerns about the health consequences of ART-conceived babies have been raised. There are reports about the association of ART with birth defects and health complications, e.g., malignancies, high blood pressure, generalized vascular functional disorders, asthma and metabolic disorders in later life. It has been suggested that hormonal treatment of the mother, and the artificial environment during the manipulation of gametes and embryos may cause genomic and epigenetic alterations and subsequent complications in the health status of ART-conceived babies. In the current study, we aimed to review the possible long-term consequences of different ART procedures on the subsequent health status of ART-conceived offspring, considering the confounding factors that might account for/contribute to the long-term consequences.

Composition and effects of seminal plasma in the female reproductive tracts on implantation of human embryos.

The function of seminal plasma involves acting as a transport medium for sperm and as a means of communication between the reproductive tissues of the male and female. It is also a vital factor to prime the reproductive tracts of the female for optimal pregnancy. When the reproductive tract of the female is exposed to seminal plasma, serious alterations take place, enhancing pathogen and debris clearance observed in the uterus throughout mating. It is also capable of supporting embryo growth, promoting the receptivity of the uterus, and establishing tolerance to the semi-allogenic embryo. Moreover, seminal plasma is capable of regulating the functions of several female reproductive organs and providing an ideal condition for effective embryo implantation and pregnancy. It is believed that the health state of the offspring is affected by exposure to seminal plasma. For the treatment of infertility, assisted reproductive technologies have been extensively employed. The application of seminal plasma as a therapeutic approach to enhance the development of embryo competency and rate of implantation, receptivity of endometrium, and establishment of maternal immune tolerance in cycles of ART appears possible. Herein, current knowledge on the composition of seminal plasma and the physiological roles it possesses on various parts of the female reproductive tract are summarized. Moreover, the role of seminal plasma in the development of embryos, implantation, and the following fetal growth and survival have been reviewed in this article.

Biologia futura: embryo-maternal communication via progesterone-induced blocking factor (PIBF) positive embryo-derived extracellular vesicles. Their role in maternal immunomodulation.

Paternal antigens expressed by the foetus are recognized as foreign. Therefore,-according to the rules of transplantation immunity-the foetus ought to be "rejected". However, during normal gestation, maternal immune functions are re-adjusted, in order to create a favourable environment for the developing foetus. Some of the mechanisms that contribute to the altered immunological environment, for example, the cytokine balance and NK cell function, with special emphasis on the role of progesterone and the progesterone-induced blocking factor (PIBF) will be reviewed.

Altered Immune Response and Implantation Failure in Progesterone-Induced Blocking Factor-Deficient Mice.

Earlier data suggest that progesterone-induced blocking factor (PIBF) is involved in implantation. The present study therefore aims to investigate the consequences of functional PIBF deficiency during the peri-implantation period. CD1 female mice were injected intraperitoneally with 2 µg anti-PIBF monoclonal antibody on days 1.5 and 4.5 of pregnancy. The number of implantation sites and resorption rates were recorded on day 10.5. PIBF+ decidual NK cells and B cells were detected by immunohistochemistry or immunofluorescence. Decidual and peripheral NK activity was assessed by flow cytometry. A prime PCR array was used for determining the differential expression of genes involved in lymphocyte activation and Th1 or Th2 differentiation in CD4+ and CD8+ spleen cells from pregnant anti-PIBF-treated and control mice. Anti-PIBF treatment in the peri-implantation period resulted in impaired implantation and increased resorption rates in later pregnancy. The number of PIBF+ decidual NK cells decreased, while both decidual and peripheral NK activity increased in the anti-PIBF-treated mice. B cells were absent from the resorbed deciduas of anti-PIBF-treated mice. The genes implicated in T cell activation were significantly downregulated in CD4+ and increased in CD8+ of the anti-PIBF-treated animals. The gene for IL-4 was significantly downregulated in CD4+ cells while that of IL-12A was upregulated in CD8+ cells of anti-PIBF-treated animals. These data suggest that the lack of PIBF results in an impaired T cell activation, together with Th1 differentiation and increased NK activity, resulting in implantation failure.

The effect of light exposure on the cleavage rate and implantation capacity of preimplantation murine embryos.

During assisted reproduction the embryos are subjected to light. We investigated the relationship between light exposure and the developmental- and implantation capacity of mouse embryos. In vitro cultured embryos were exposed to white or red filtered light, then transferred to the uteri of pseudo-pregnant females. The mice were sacrificed on day 8.5 and implantation sites were counted. The number of nucleic acid containing (PI+) extracellular vesicles (EVs) in culture media of light-exposed and control embryos, as well as, the effect of the EVs on IL-10 production of CD8+ spleen cells was determined by flow cytometry. DNA fragmentation in control and light exposed embryos was detected in a TUNEL assay. The effect of light on the expression of apoptosis-related molecules was assessed in an apoptosis array. Light exposure significantly reduced the implantation capacity of the embryos. The harmful effect was related to the wavelength, rather than to the brightness of the light. Culture media of light exposed groups contained significantly higher number of PI + EVs than those of the control embryos, and failed to induce IL-10 production of spleen cells. The number of nuclei with fragmented DNA, was significantly higher in embryos treated with white light, than in the other two groups. In conclusion exposure to white light impairs the implantation potential of in vitro cultured mouse embryos. These effects are partly corrected by using a red filter. Since there is no information on the light sensitivity of human embryos, embryo manipulation during IVF and ICSI should be performed with caution.

PIBF+ extracellular vesicles from mouse embryos affect IL-10 production by CD8+ cells.

Earlier evidence suggests, that the embryo signals to the maternal immune system. Extracellular vesicles (EVs) are produced by all types of cells, and because they transport different kinds of molecules from one cell to the other, they can be considered as means of intercellular communication. The aim of this work was to test, whether the embryo is able to produce sufficient amounts of EVs to alter the function of peripheral lymphocytes. Embryo-derived EVs were identified by their Annexin V biding capacity, and sensitivity to Triton X dependent lysis, using flow cytometry. Transmission electron microscopy was used to detect EVs at the implantation site. Progesterone-induced blocking factor (PIBF) expression in embryo-derived EVs was demonstrated with immuno-electron microscopy. The % of IL-10 + murine lymphocytes was determined by flow cytometry. EVs were present in embryo culture media, but not in empty media. Mouse embryo-derived EVs adhere to the surface of both CD4+ and CD8+ murine peripheral T lymphocytes, partly, via phosphatidylserine binding. The number of IL-10+ murine peripheral CD8+ cells increases in the presence of embryo-derived EVS, and this effect is counteracted by pre-treatment of EVs with an anti-PIBF antibody, suggesting that the embryo communicates with the maternal immune system via EVs.

A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer.

Multiple pregnancy is a risk for prematurity and preterm birth. The goal of assisted reproduction is to achieve a single pregnancy, by transferring a single embryo. This requires improved methods to identify the competent embryo. Here, we describe such a test, based on flow cytometric determination of the nucleic acid (PI+) containing extracellular vesicle (EV) count in day 5 embryo culture media. 88 women undergoing IVF were included in the study. More than 1 embryos were transferred to most patients. In 58 women, the transfer resulted in clinical pregnancy, whereas in 30 women in implantation failure. In 112 culture media of embryos from the "clinical pregnancy" group, the number of PI+ EVs was significantly lower than in those of 49 embryos, from the "implantation failure" group. In 14 women, transfer of a single embryo resulted in a singleton pregnancy, or, transfer of two embryos in twin pregnancy. The culture media of 19 out of the 20 "confirmed competent" embryos contained a lower level of PI+ EVs than the cut off level, suggesting that the competent embryo can indeed be identified by low PI+ EV counts. We developed a noninvasive, simple, inexpensive, quick test, which identifies the embryos that are most likely to implant.

The decidua-the maternal bed embracing the embryo-maintains the pregnancy.

The decidua has been known as maternal uterine tissue, which plays essential roles in protecting the embryo from being attacked by maternal immune cells and provides nutritional support for the developing embryo prior to placenta formation. However, there are questions that still remain to be answered: (1) How does the decidua supply nutrition and provide a physical scaffold for the growing embryo, before placental vascular connection is established? (2) How is the balance between preventing an anti-embryo immune response and protecting both embryo and mother from infections established? To understand basic personas in decidual tissues, we review the structure of the decidua composed of terminally differentiated uterine stromal cells, blood vessels, and a number of repertoire of uterine local immune cells, including the well-known uterine natural killer (uNK) cells and recently discovered innate lymphoid cells (ILCs). Decidual macrophages and uterine dendritic cells (DCs) are supposed to modulate adaptive immunity via balancing cytokines and promoting generation of regulatory T (Treg) cells. During decidualization, vascular and tissue remodeling in the uterus provide nutritional and physical support for the developing embryo. Secretion of various cytokines and chemokines from both the embryo and the decidual cells activates multiple signaling network between the mother and the embryo upon implantation. Defects in the decidual development during early pregnancy result in loss of pregnancy or complications in later gestational stage.