The evaluation of PD-1 and Tim-3 expression besides their related miRNAs in PBMCs of women with recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a multifactorial disorder, associated with immunologic abnormalities. During pregnancy, the maternal immune system uses different tolerance mechanisms to deal with a semi-allogenic fetus. The expression of immune checkpoints and their related miRNAs in immune cells can ensure pregnancy at the feto-maternal interface by modulating immune responses. This study aims to evaluate the expression of the immune checkpoint molecules PD-1 and Tim-3 on circulating T cells by flow cytometry, that of mir-138 and mir-155 in PBMCs by Real-time PCR, and the concentrations of TGF-ß and IP-10 in the sera of women suffering from RPL as well as of gestational age-matched healthy pregnant women by ELISA. The percentage of PD-1 or Tim-3 expressing CD8+ T cells was significantly lower in RPL patients compared to the controls, while there was no significant difference in Tim-3 expression of CD4+ T cells between the two groups. The mRNA of both the PD-1 and Tim-3 genes were downregulated in PBMCs of RPL patients compared to controls, however, the difference was not statistically significant for Tim-3. The concentration of TGF-ß was significantly lower and that of IP-10 was significantly higher in the sera of RPL patients than in those of the controls. The relative expression of mir-138 and miR-155 were significantly lower, in PBMCs of RPL patients than in those of healthy pregnant women. These data confirm that by affecting cytokine production, immune checkpoints, and microRNAs play a role in establishing the appropriate local immune environment for successful pregnancy. The wider analysis of immune checkpoints may also yield new biomarkers for the diagnosis and prevention of RPL.

Clinical Benefits of Decreased Photo-Oxidative Stress on Human Embryo Development.

OBJECTIVE: Early embryonic development is characterized by rapid cell division and gene activation, making the embryo extremely sensitive to environmental influences. Light exposure can affect embryonic development through a direct toxic effect on the embryo via the generation of reactive oxygen species. In a previous study, we demonstrated the positive effect of improved light-protected embryo culture conditions implemented in our laboratory. This study aimed to investigate the changes in human embryo development under light protection during the conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: We tested the potential beneficial effect of light filters to reduce the risk of toxic effects of light. IVF outcomes were compared between two experimental conditions, light protection with red light filters versus no light protection as a control. RESULTS: Blastocyst development rate in IVF was significantly higher in the light-protected group than in the group treated under conventional conditions (46.6 vs. 26.7%). In the case of ICSI, we obtained a similar result (44.5 vs. 31.6%). The rate of cryopreservation with at least one embryo was higher in the light-protected phase (32.8%) than in the conventionally manipulated phase (26.8%). The abortion rate was also significantly lower during the light-protected period in IVF, resulting in a higher live birth rate. CONCLUSIONS: The implementation of light protection to reduce the embryotoxic wavelengths of light in IVF centers may improve the blastocyst development rate and embryo quality while maintaining embryo safety.

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.

The effect of calcitriol on the development and implantation capacity of embryos from hyper-stimulated mice.

The evidence concerning the role of vitamin D (VD) in reproduction is still inconclusive. Calcitriol was given to superovulated female mice at the time of FSH injection (Group A), or at day 0.5 of pregnancy (Group B). The retrieved and cultured embryos were transferred to the uteri of pseudopregnant females. Ten animals from each group conceived naturally, and at day 7.5 of pregnancy, the implantation sites were counted. Serum hormone concentrations were determined by ELISA. The expression of CD70, PD-L1, OX-40L, and PIBF on extracellular vesicles (EVs) was tested by flow cytometry. Calcitriol treatment did not alter serum oestradiol concentrations, while 25(OH) D levels significantly decreased in both treated groups. Progesterone concentrations were significantly higher in group A and lower in group B than in the controls. On EVs produced by group B embryos PIBF, CD70, and OX-40L expression were significantly lower, while that of PD-L1 was significantly higher than that of controls. Calcitriol treatment decreased the fertilization rate in group A, and the blastulation rate of cultured embryos in group B, while the implantation capacity of the embryos was not affected, suggesting that depending on the time of administration, VD has an adverse effect on oocyte maturation and embryo development, but not on the implantation rates.

The two faces of preeclampsia / A praeeclampsia két arca

During normal pregnancy, blood volume increases by nearly two liters. Distinctively, the absence and also the extreme extent regarding the volume expansion are likely accompanied with serious conditions. Undoubtedly, preeclampsia, defined as the appearance of hypertension and proteinuria during the second half of pregnancy, is not a homogenous disease. The early onset which begins prior to the 34th week, is characteristically a hypovolemia-associated form and depicts the placental origination, in which endothelial damage leads to hypertension and organ damage due to vasoconstriction and microthrombosis. Fetal blood supply progressively worsens due to placental insufficiency. The outcome of this condition often leads to fetal death, eclampsia, or placental abruption. Management is confined to a diligent prolongation of pregnancy to accomplish improved neonatal pulmonary function. The late onset form, associated with high cardiac output, is a maternal disease, in which obesity is a risk factor since it predisposes individuals to enhanced water retention, hypertension, and a weakened endothelial dysfunction. Initially, low extremity edema often times progresses to a generalized form and frequently results in hypertension. In several cases proteinuria appears. This condition entirely meets the preedampsia criteria. Fetal weight is normal or frequently over the average. It is very likely, the increasing parenchymal stasis will lead to ascites, eclampsia, or placental abruption. During the management of this hypervolemia-associated preedampsia, the administration of diuretic furosemide treatment seemingly offers promise.

Progesterone: A Unique Hormone with Immunomodulatory Roles in Pregnancy.

Progesterone is well known for its numerous endocrinologic roles in pregnancy but is also endowed with fascinating immunomodulatory capabilities. It can downregulate the induction of inflammatory reactions, the activation of immune cells and the production of cytokines, which are critical mediators of immune responses. These features appear to be critical to the success of pregnancy, given the ability of maternal immune reactivity to interfere with pregnancy and to contribute to several pregnancy complications. This review summarizes the contribution of maternal immune effectors in general, and cytokines in particular, to pregnancy complications such as recurrent miscarriage, pre-eclampsia and preterm labor; it describes the promise offered by supplementation with progesterone and the oral progestogen dydrogesterone, as well as the progesterone-induced blocking factor in the prevention and/or treatment of these serious complications.

Adverse effects on female fertility from vaccination against COVID-19 unlikely.

This opinion paper briefly presents arguments that support the unlikelihood of an impact on female fertility from current covid-19 vaccines.

Key to Life: Physiological Role and Clinical Implications of Progesterone.

The most recent studies of progesterone research provide remarkable insights into the physiological role and clinical importance of this hormone. Although the name progesterone itself means "promoting gestation", this steroid hormone is far more than a gestational agent. Progesterone is recognized as a key physiological component of not only the menstrual cycle and pregnancy but also as an essential steroidogenic precursor of other gonadal and non-gonadal hormones such as aldosterone, cortisol, estradiol, and testosterone. Based on current findings, progesterone and novel progesterone-based drugs have many important functions, including contraception, treatment of dysfunctional uterine bleeding, immune response, and prevention of cancer. Considering the above, reproduction and life are not possible without progesterone; thus, a better understanding of this essential molecule could enable safe and effective use of this hormone in many clinical conditions.

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.

Cytokines, Hormones and Cellular Regulatory Mechanisms Favoring Successful Reproduction.

Its semi-allogeneic nature renders the conceptus vulnerable to attack by the maternal immune system. Several protective mechanisms operate during gestation to correct the harmful effects of anti-fetal immunity and to support a healthy pregnancy outcome. Pregnancy is characterized by gross alterations in endocrine functions. Progesterone is indispensable for pregnancy and humans, and it affects immune functions both directly and via mediators. The progesterone-induced mediator - PIBF - acts in favor of Th2-type immunity, by increasing Th2 type cytokines production. Except for implantation and parturition, pregnancy is characterized by a Th2-dominant cytokine pattern. Progesterone and the orally-administered progestogen dydrogesterone upregulate the production of Th2-type cytokines and suppress the production of Th1 and Th17 cytokine production in vitro. This is particularly relevant to the fact that the Th1-type cytokines TNF-α and IFN-γ and the Th17 cytokine IL-17 have embryotoxic and anti-trophoblast activities. These cytokine-modulating effects and the PIBF-inducing capabilities of dydrogesterone may contribute to the demonstrated beneficial effects of dydrogesterone in recurrent spontaneous miscarriage and threatened miscarriage. IL-17 and IL-22 produced by T helper cells are involved in allograft rejection, and therefore could account for the rejection of paternal HLA-C-expressing trophoblast. Th17 cells (producing IL-17 and IL-22) and Th22 cells (producing IL-22) exhibit plasticity and could produce IL-22 and IL-17 in association with Th2-type cytokines or with Th1-type cytokines. IL-17 and IL-22 producing Th cells are not harmful for the conceptus, if they also produce IL-4. Another important protective mechanism is connected with the expansion and action of regulatory T cells, which play a major role in the induction of tolerance both in pregnant women and in tumour-bearing patients. Clonally-expanded Treg cells increase at the feto-maternal interface and in tumour-infiltrating regions. While in cancer patients, clonally-expanded Treg cells are present in peripheral blood, they are scarce in pregnancy blood, suggesting that fetal antigen-specific tolerance is restricted to the foeto-maternal interface. The significance of Treg cells in maintaining a normal materno-foetal interaction is underlined by the fact that miscarriage is characterized by a decreased number of total effector Treg cells, and the number of clonally-expanded effector Treg cells is markedly reduced in preeclampsia. In this review we present an overview of the above mechanisms, attempt to show how they are connected, how they operate during normal gestation and how their failure might lead to pregnancy pathologies.

How to Reduce the Potential Harmful Effects of Light on Blastocyst Development during IVF.

PURPOSE: Earlier findings revealed the damaging effect of visible light on zygotes and gametes. The aim of our study is to eliminate or significantly reduce the potentially harmful effects of light exposure during in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and to investigate the effect of light protection on embryo development and implantation. MATERIALS AND METHODS: To protect sperm cells, oocytes, and embryos from the potential harmful effects of light exposure during laboratory procedures, we created a dark environment for the cells and applied red filters on laboratory lamps and UV or infrared filters in the microscopes in order to eliminate white light exposure of the cells throughout all work stages. RESULTS: The fertilization rate was significantly (p = 0.011) higher in light-protected ICSI cycles. Blastocyst development rates (blastocyst/embryo) were significantly (p < 0.001) higher in light-protected embryos than in those manipulated in conventional light conditions both in IVF (20.9% difference) and ICSI (38.6% difference). Numbers of clinical pregnancies/transfers of ICSI fertilized day 5 blastocysts were also significantly (p = 0.040) higher in light-protected conditions. CONCLUSIONS: These data show that light protection has a positive effect on fertilization rate and increases the blastocyst development as well as the number of clinical pregnancies/transfers. Implementation of this light protection method in IVF centers may improve the success rate while maintaining maximal embryo safety.

Spliceosome protein EFTUD2 is upregulated in the trophoblast of spontaneous miscarriage and hydatidiform mole.

BACKGROUND: Elongation factor Tu GTP binding domain containing 2 (EFTUD2) is an alternative splicing factor that modulates cell differentiation and activation processes. EFTUD2 is known to modulate immune responses and mutation of the EFTUD2-gene lead to fetal malformation. Little is known about its expression and role in normal and disturbed first trimester pregnancy. PATIENTS AND METHODS: We investigated the expression of EFTUD2 in placental tissue obtained from patients with normal (n = 14), spontaneous miscarriage (n = 15) and molar (n = 14) pregnancy by immunohistochemistry. The expression of EFTUD2 was correlated on the protein level with known immune modulatory proteins like pregnancy zone protein (PZP) and in addition with human chorionic gonadotropin (hCG). Furthermore, we analysed the EFTUD2 and PZP expression in vitro after stimulation of the chorioncarcinoma cell line JEG-3 with hCG. RESULTS: EFTUD2 is significantly upregulated in the syncytiotrophoblast of spontaneous miscarriage (p = 0.003) and molar pregnancy (p = 0.003) compared to week of gestation-adjusted normal first trimester placentas. PZP is negatively correlated (p = 0.021) to EFTUD2 in the syncytiotrophoblast and is therefore significantly downregulated in miscarriage (p = 0.028) and mole pregnancy (p = 0.006). In addition, hCG is positively correlated to EFTUD2 in mole pregnancy. The addition of hCG to chorioncarcinoma cell lines JEG-3 in vitro stimulated EFTUD2 expression in these cells (p = 0.027). CONCLUSION: Regulation of alternative splicing seems crucial for a successful ongoing pregnancy. The up-regulated elongation factor EFTUD2 may have a critical role in miscarriage.

Progesterone induced blocking factor (PIBF) taken in early pregnancy predicts the pregnancy outcome in women undergoing in vitro fertilization procedure.

Earlier data suggest a relationship between PIBF concentrations and the outcome of pregnancy. The aim of the study was to compare serum and urine concentrations of PIBF in women with successful pregnancy after IVF with those of women without pregnancy after IVF procedure, and to evaluate the potential relation between PIBF and the outcome of pregnancy. Urine and serum were collected from 120 women, undergoing IVF. 87.5% of patients had primary infertility. 69.2% faced female causes of infertility: 10.8% tubal cause, 11.7% ovulation disorder, and 46.7% other causes of infertility. 30.8% of patients had male factor of infertility. Among non-pregnant women (42) mean concentrations of PIBF in urine and serum were significantly lower (15.8 ng/mL; 148.4 ng/mL) than in women with positive beta HCG value (78) (19.1 ng/mL; 225.9 ng/mL). In 49 patients pregnancy terminated with a term delivery, in 10 patients with pretem delivery, while in 19 patients the pregnancy terminated with a miscarriage. PIBF concentrations in urine (13.9 ± 2.8 ng/mL) and serum (124.6 ± 46.7 ng/mL) samples of women with miscarriage were significantly lower of those with preterm delivery (180.6 ± 54.4 ng/mL; 18.1 ± 4.4 ng/mL) and of those with term delivery (20.4 ± 8.5 ng/mL; 208.7 ± 114.3 ng/mL). Successful pregnancy after IVF procedure is predictable by measuring of urine and serum PIBF concentrations and could be important for predicting of early implantation and pregnancy outcome after IVF procedure and maybe to protect the risk pregnancy.

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.

The involvement of the progesterone receptor in PIBF and Gal-1 expression in the mouse endometrium.

PROBLEM: The progesterone-regulated genes, PIBF and Gal-1, are key players in the feto-maternal immunological interaction. This study aims to investigate the expression of PIBF and Gal-1 in WT and progesterone receptor KO models as well as subsequent effects of PIBF on decidualization of stromal cells. METHOD OF THE STUDY: PRAKO, PRBKO and PRKO BALB/c mice were used for assessing the role of PR isoforms in PIBF induction. PIBF- and Gal-1 mRNA expression in the uterus was tested by real-time PCR. The effect of PIBF on decidualization of endometrial stromal cells was verified by anti-desmin immunofluorescence. Immunohistochemistry was used for testing PIBF expression in the uterus. Gal-1, ERα and PR positive decidual NK cells were detected by immunofluorescence. RESULTS: PIBF mRNA was significantly increased in progesterone-treated WT mice, but not in PRKO and PRAKO mice. PIBF protein expression was reduced in the endometria of PRKO and PRAKO, but not in PRBKO mice. During a 6-day culture, PIBF induced decidual transformation of endometrial stromal cells. PIBF expression in the mouse uterus was highest during the implantation window, while Gal-1 mRNA expression continuously increased between day 2.5 and day 11.5 of gestation. Decidual NK cells express Gal-1 and ERα, but not PR at day 7.5 murine pregnancy. CONCLUSION: PIBF produced via engagement of PRA, is highly expressed in the endometrium during the implantation window, and plays a role in decidualization. The concerted action of PIBF and Gal-1 might contribute to the low cytotoxic activity of decidual NK cells.

The Role of Progesterone in Feto-Maternal Immunological Cross Talk.

This review aims to provide a brief historical overview of the feto-maternal immunological relationship, which profoundly influences the outcome of pregnancy. The initial question posed in the 1950s by Medawar [Symp Soc Exp Biol. 1953; 7: 320-338] was based on the assumption that the maternal immune system recognizes the fetus as an allograft. Indeed, based on the association between HLA-matching and spontaneous miscarriage, it became obvious that immunological recognition of pregnancy is required for a successful gestation. The restricted expression of polymorphic HLA antigens on the trophoblast, together with the presence of nonpolymorphic MHC products, excludes recognition by both T and NK cells of trophoblast-presented antigens; however, γδ T cells, which constitute the majority of decidual T cells, are likely candidates. Indeed, a high number of activated, progesterone receptor-expressing γδ T cells are present in the peripheral blood of healthy pregnant women and, in the presence of progesterone, these cells secrete an immunomodulatory protein called progesterone-induced blocking factor (PIBF). As early as in the peri-implantation period, the embryo communicates with the maternal immune system via PIBF containing extracellular vesicles. PIBF contributes to the dominance of Th2-type reactivity which characterizes normal pregnancy by inducing increased production of Th2 cytokines. The high expression of this molecule in the decidua might be one of the reasons for the low cytotoxic activity of decidual NK cells.

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.

The effect of the Progesterone-Induced Blocking Factor (PIBF) on E-cadherin expression, cell motility and invasion of primary tumour cell lines.

In addition to being immunomodulatory, Progesterone-Induced Blocking Factor (PIBF) plays a role in cell cycle regulation and invasion. The full length protein is associated with the pericentriolar satellites and as such, it is crucial for maintaining the integrity of spindle poles during mitosis. Another suggestive evidence for the involvement of PIBF in tumour progression is the fact that the PIBF gene has been identified on chromosome 13 in the region associated with breast cancer susceptibility. Earlier we showed that PIBF differentially regulates the invasiveness of trophoblast and tumour cell lines. The aim of the present study was to further investigate the role of PIBF in tumour development, using primary ovarian- (OC) and primary lung carcinoma (LC) cell cultures, and JEG-3 choriocarcinoma cell line. In the cultured cells PIBF was knocked down by siRNA treatment, and the impact of PIBF deficiency on MMP-9 activity and E-cadherin expression as well as on invasive and migratory capacity of the cells was tested. In conditioned media of PIBF-deficient JEG-3 cells, LC cells and OC cells MMP-9 activity was reduced to 36% 35%, and 65% respectively compared to controls. Though PIBF knock down did not affect migration, in JEG-3 cells, LC primary cells and OC primary cells PIBF deficiency resulted 20%, 50% and 50% decrease of invasion respectively. PIBF silencing resulted in increased E-cadherin expression, suggesting that by down regulating E-cadherin expression, PIBF might interfere with the cell-cell adhesion mechanisms and by increasing MMP activity induced extracellular matrix degradation, facilitates the invasion of tumour cells.