The research was concentrated on dynamical gene expression profiling upon pathological changes ultimately leading to CC. Differences of normalised mRNA concentrations were used for quantitative model design and its primary approbation.

No abstract

The expression of immune response gene mRNA in the umbilical and venous blood were compared in newborns of the first day of life with and without signs of infection. The expression of il1b, il6, il8, il10, il12a, il15, il18, tnfa, tgfb1, tbx21, gata3, foxp3, rorc2, cd45, cd68, cd69, tlr2, tlr4, tlr9, and mmp8 mRNA was evaluated in umbilical and venous blood cells of newborns by reverse transcription real time PCR. In full-term newborns without signs of infection, the expression of il8, tlr2, tlr4, and mmp8 in venous blood was higher than in umbilical blood, while in preterm newborns, the levels of mmp8 transcript were elevated while the levels of tlr9, cd45, and gata3 were reduced. The expression of some markers differed in the umbilical and venous blood and in newborns with congenital infectious disease and without signs of infection.

Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

Study question How does the woman’s reproductive age affect the ability of follicular fluid (FF) extracellular vesicles (EV) to change the motility and hyperactivation of spermatozoa and follicular fluid microRNA profiles? Summary answer The effect of FF EVs on sperm motility and hyperactivation decreases with a woman's age. MiR-134-5p, miR-21-5p expression levels increase in older age group. What is known already Aging reduces human fertility. FF is an important factor in attracting and activating spermatozoa in the oviduct during oocyte fertilization, enhancing the process of sperm capacitation and acrosomal reaction in various mammalian species, including humans. EVs from seminal plasma and oviduct fluid carry proteins and miRNAs playing a vital role in a multi-step process including sperm motility, capacitation, acrosomal reaction, further fertilization. Results of our previous experiments showed that FF EVs from young women significantly improve the indices of sperm motility and hyperactivation. Several studies have isolated some age associated differentially expressed miRNAs (hsa-miR-424, hsa-miR-21-5p, hsa-miR-134, hsa-miR-190b, hsa-miR-99b -3p). Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds, frozen at -80 °C. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 106/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Samples were centrifuged and fixed in 2.5% glutaraldehyde in 0.1 M buffer for TEM. Sperm motility were assessed using CASA. MicroRNAs isolation (miRNeasy Serum/Plasma Kit (Qiagen)). Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 18) aged 28-36 years without severe pathozoospermia. FF EVs were obtained from young (n = 4) aged 27-31 (EVs+) and older patients (n = 4) aged 41-46 with several IVF attempts (EVs-). The methods used in this work include sperm and EVs isolation, incubation, transmission electron microscopy, RT-PCR, statistical data analysis, computer-assisted semen analysis (CASA). Main results and the role of chance Our results showed that sperm incubation with EVs+ led to a significant increase in the number of progressively motile spermatozoa (paired Student's t test, p < 0.001), indicators of general mobility (p = 0.05) and hyperactivation of spermatozoa after 1 hour. Sperm incubation with EVs- slightly changed these parameters compared to the control (p = 0.171). Results from TEM showed that EVs- bind to spermatozoa worse than EVs+ after incubation. EVs- bind predominantly to the lateral membrane of the spermatozoon, rather than to the acrosomal region, compare to EVs+, which may reflect changes in the functional composition of FF EVs in older patients group and target areas of interaction with spermatozoa. This hypothesis is also supported by slight improvements in progressive sperm motility after incubation. Age-related changes are reflected in energy, metabolic and other important biological processes, which leads to a change in the composition and functions of FF (decrease in progesterone, glucose, increase in lactate and pyruvate, increase in reactive oxygen species) and affect the functional role of FF EVs. MiR-134-5p (p = 0.008), miR-21-5p (p = 0.008) expression levels were statistically significantly higher in EVs- group compared with EV +  group with a successful first attempt. The expression of other microRNAs analyzed did not differ between groups. Limitations, reasons for caution Small amount of data. This study does not characterize isolated FF EVs in any way (specific markers, EVs concentration, size). To discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics, it is necessary to perform metabolomics analyses of FF EVs. Wider implications of the findings Further studies of the influence of FF EVs on sperm motility may help to improve of ART outcomes, associated with male and female infertility by improving sperm morphofunctional characteristics and increasing their fertilizing ability. The solutions to this problem could be the use of FF EVs from young fertile donors. Trial registration number not applicable