Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.

Cutting-edge techniques provide insights regarding repeated implantation failure patients.

RESEARCH QUESTION: Can time-lapse parameters and the transcriptional profile of cumulus cells be used to achieve a more stringent and non-invasive method of embryo assessment and to identify possible factors affecting the embryo's ability to implant in repeated implantation failure (RIF) patients? DESIGN: A total of 190 embryos from 18 oocyte donors and 145 embryos from 15 RIF patients were evaluated based on time-lapse parameters. Three morphokinetic parameters including T5 (time to reach five cells), T3 (time to reach three cells) and CC2 (time to two to three cells) were recorded for all embryos. Embryos that had all three parameters in the normal range were graded as high quality and comparison between these parameters were compared in high-quality embryos between two groups. The transcriptional profile of cumulus cells related to high-quality embryos of both groups were analysed by RNA sequencing and compared. Finally, the possible relationship between differentially expressed genes and time-lapse parameters was examined. RESULTS: T5 was significantly lower in the RIF group than the donor group (P = 0.011). The cumulus cell transcriptome analysis showed 193 genes were down-regulated and 222 genes up-regulated. The mammalian target of rapamycin and the transforming growth factor beta pathways were significantly increased in the RIF group compared to the donor group (P = 0.007 and 0.01, respectively). Vitamin B12 and fatty acid beta-oxidation pathways were also significantly reduced in the RIF group compared to the donor group (P = 0.006 and 0.01, respectively). CONCLUSIONS: Differences in the transcriptomic profiles of cumulus cells and some morphokinetic parameters may be one of the main factors contributing to unexplained RIF.

MiRNAs secreted by human blastocysts could be potential gene expression regulators during implantation.

BACKGROUND: Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs are secreted by a blastocyst in culture media. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs as well as other soluble factors and regulate their receptivity-related gene expression. METHODS AND RESULTS: Blastocyst culture media (BCM) were collected from the individually cultured embryos, while human endometrial epithelial cells (HEECs) were collected from healthy fertile volunteers. To evaluate the effect of BCM on the endometrial receptivity gene expression, HEECs were co-cultured with implanted BCM, non-implanted BCM, and a control culture medium. After determining altered gene expression in the HEECs, the miRNAs-related genes through bioinformatics databases were identified and evaluated in the BCM. Co-culture of primary HEECs with BCM significantly stimulated the expression levels of VEGFA, HBEGF, HOXA10, and LIF in the implanted group compared with non-implanted and control groups. The fold changes of miR-195 significantly diminished in the implanted BCM group compared with the non-implanted BCM group. Reduced fold changes of miR-29b, 145 and increased miR-223 were also observed in the implanted BCM group compared with the non-implanted ones. CONCLUSION: miRNAs could function as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, taken up by endometrial epithelial cells, and cause a change in the endometrial function. We found that BCMs can be effective in implantation process by stimulating related receptivity gene expression.

Effects of myo-inositol plus folic acid on ovarian morphology and oocyte quality in PCOS mouse model.

Although the role of myo-inositol (MYO) in promoting the oocyte quality of PCOS patients has been documented in human studies; the cellular effects of this supplement on oocytes have not been directly examined due to ethical limitations. In the first phase of this study, MYO dosimetry was carried out simultaneously with the PCOS model development. An effective dose was obtained following the assessment of fasting insulin and testosterone levels using ELISA and ovarian morphology appraisal by histopathology. In the second phase, following the continuous administration of the effective dose of MYO and dehydroepiandrosterone (DHEA), cellular evaluation was performed. The quality of oocytes from superovulation was analyzed by examining maturity and normal morphology percentage using a stereomicroscope, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels using fluorometry, and ATP count evaluation using ELISA. The results revealed that, among the four different MYO concentrations, the 0.36 mg/g dose compared with the DHEA group reduced testosterone levels and large atretic antral follicles (LAtAnF) diameter. This dose also increased the corpus luteum count and the granulosa:theca (G/T)layer thickness ratio in antral follicles. Furthermore, this dose increased mature oocytes and normal morphology percentage, ATP count, and GSH levels; however, it decreased ROS levels in mature oocytes. Our findings provide the grounds for further cellular and molecular studies on the PCOS mouse model, suggesting that the improvement in mitochondrial function and its antioxidant properties is probably one of the mechanisms by which MYO increases oocyte quality.

The effect of Myo-Inositol supplement on molecular regulation of folliculogenesis, steroidogenesis, and assisted reproductive technique outcomes in patients with polycystic ovarian syndrome.

RESEARCH QUESTION: The mechanism of Myo-Inositol, as an adjuvant, on key signaling pathways related to oocyte maturation, fertilization rate, and embryo quality as well as ovarian steroidogenesis in cumulus cells of PCOS patients, is still unclear. DESIGN: Infertile patients who were candidates for ART cycles were divided into three groups (n = 30 in each group), including group 1: PCOS patients only receiving folic acid, group 2: PCOS patients receiving daily Myo-Inositol combined with folic acid, and a control group (group 3): normal ovulatory women without PCOS receiving only folic acid from 1 month prior to IVF cycle until the day of ovum pick up. During the ART procedure, oocytes maturation, fertilization rate, and embryo quality were assessed. The gene expressions of FSHR, LHR, CYP11A1, CYP19A1, 3ß-HSD2, and StAR were also analyzed using qRT-PCR. Western blot analysis was performed for the evaluation of AKT, ERK, CREB, and AMPK phosphorylation. RESULT: Despite equal number of retrieved oocytes, the percentages of MII oocytes, fertilization rate, and embryo quality were found to be significantly higher in group 2 due to the administration of inofolic. The expressions of all the studied genes were significantly higher in the cumulus cells of group 1 compared to the group 2. Higher phosphorylation of ERK1/2 was found in the groups 2 and 3 compared to the group 1. On the other hand, p-Akt has significantly decreased in the group 2 compared to the group 1. CONCLUSION: Our study provides new insight into the molecular mechanism underlying the positive effect of Myo-Inositol on intrinsic ovarian defects in PCOS, steroidogenesis, oocyte maturation, fertilization rate, and embryo quality.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

Microfluidic chips as a method for sperm selection improve fertilization rate in couples with fertilization failure.

PURPOSE: Sperm quality plays a vital role in successful fertilization and pregnancy. Patients with fertilization failure (total failure or low-fertilization rate) despite having normal semen parameters are a challenging group whose sperm cannot fertilize the oocyte via the intracytoplasmic sperm injection (ICSI) technique. Microfluidics is offered as a new method for proper sperm sorting. METHODS: This study aimed to evaluate sperm parameters, DNA fragmentation index (DFI), expression of phospholipase C zeta 1 (PLCZ1), and transition nuclear proteins 1 (TNP1) mRNAs in sperm selected by microfluidic sperm sorting (MSS) chip compared with conventional density gradient centrifugation technique in patients with fertilization failure following ICSI. Subsequence fertilization rate and embryo quality were assayed. RESULTS: Normal morphology and total motility were significantly higher, and DFI was significantly lower in sperm selected by the MSS chip in fertilization failure and control groups. The RT-PCR results demonstrated a significant increase in the expression of PLCZ1 and TNP1 genes in sperm of both groups selected by MSS chips compared to the DGC method. In addition, with the selected sperm by MSS chip, an increase in fertilization rate and improvement of embryo quality was obtained. CONCLUSION: The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

The myo-inositol effect on the oocyte quality and fertilization rate among women with polycystic ovary syndrome undergoing assisted reproductive technology cycles: a randomized clinical trial.

PURPOSE: The aim of the present study was to evaluate the effect of myo-Inositol administration on oocyte quality, fertilization rate and embryo quality in patients with PCOS during assisted reproductive technology (ART) cycles. METHODS: Fifty infertile PCOS patients were randomly designated in two groups. In the study group, patients received daily doses of 4 g myo-Inositol combined with 400 mg folic acid and in the control group patients received only 400 mg folic acid from 1 month before starting the antagonist cycle until the day of ovum pick up. Oocyte and embryo qualities were assessed according to European Society of Human Reproduction and Embryology (ESHRE) guidelines. The gene expression of PGK1, RGS2 and CDC42 as a factor of oocyte quality in granulosa cells was analyzed using real-time RT-PCR. Levels of total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated by chemiluminescence assay in follicular fluid. RESULTS: The percentage of metaphase II oocyte, fertilization rate and embryo quality significantly improved in the study group (p < 0.05), but the number of retrieved oocytes and follicle count were not statistically different between groups. Furthermore, the gene expression of PGK1, RGS2 and CDC42 was significantly higher in the study group (p < 0.05) but no differences were found between two groups in terms of TAC and ROS levels. CONCLUSIONS: The present study findings suggest that myo-Inositol alters the gene expression in granulosa cells and improves oocyte and embryo quality among PCOS patients undergoing ART.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

RESEARCH QUESTION: What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? DESIGN: In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. RESULTS: Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. CONCLUSIONS: The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Apolipoprotein A1 as a novel anti-implantation biomarker in polycystic ovary syndrome: A case-control study.

BACKGROUND: Women with polycystic ovary syndrome have lower pregnancy rates, possibly due to the decreased uterine receptivity. Successful implantation depends on protein networks that are essential for cross-talk between the embryo and endometrium. Apolipoprotein A1 has been proposed as a putative anti-implantation factor. In this study, we evaluated apolipoprotein A1 expression in human endometrial tissues. MATERIALS AND METHODS: Endometrial apolipoprotein A1 messenger RNA (mRNA) and protein expression were investigated using quantitative real-time polymerase chain reaction (PCR) and Western blot. The distribution of apolipoprotein A1 was also detected by immunostaining. Samples were obtained from 10 patients with polycystic ovary syndrome and 15 healthy fertile women in the proliferative (on day 2 or day 3 before ovulation, n = 7) and secretory (on days 3-5 after ovulation, n = 8) phases. RESULTS: Endometrial apolipoprotein A1 expression was upregulated in patients with polycystic ovary syndrome compared to normal subjects. However, apolipoprotein A1 expression in the proliferative phase was significantly higher than in the luteal phase (P value < 0.05). CONCLUSION: It seems that differentially expressed apolipoprotein A1 negatively affects endometrial receptivity in patients with polycystic ovary syndrome. The results showed that apolipoprotein A1 level significantly changes in the human endometrium during the menstrual cycle with minimum expression in the secretory phase, coincident with the receptive phase (window of implantation). Further studies are required to clarify the clinical application of this protein.

How sperm protects itself: A journey in the female reproductive system.

Sperm must pass a complex route in the female reproductive tract (FRT) to reach the fertilization site and join the oocyte. Thus, it should employ several mechanisms to survive against the female immune system, fertilize the oocyte, and successfully transmit paternal genes to the next generation. In addition to self-protection, sperm may be involved in the immune tolerance to the developing embryo and regulating the FRT for embryo implantation and subsequent pregnancy. Hence, this review intends to summarize the mechanisms that protect sperm in the FRT: including immunomodulatory factors that are carried by seminal plasma, cell-to-cell and molecular interaction of sperm with epithelial and immune cells of the FRT, high regulated secretions of inflammatory factors such as cytokines, chemokines, and growth factors, inducing immune tolerance to paternal antigens, and specialized expression of cell receptors and binding proteins. In most of these events sperm induces the FRT to protect itself by modulating immune responses for its own benefit. However, not all sperm in the semen are able to trigger the survival mechanisms and only high-quality sperm will overcome this challenge. A clear understanding of the molecular mechanisms that maintain sperm viability and function in the FRT can lead to new knowledge about infertility etiology and a new approach in assisted reproductive technologies for the preparation and selection of the best sperm based on the criteria that physiologically happen in-vivo.

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 µL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

Advanced strategies for single embryo selection in assisted human reproduction: A review of clinical practice and research methods.

Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.

Metabolomic profiling of embryo culture media in patients with repeated implantation failure during assisted reproductive technology cycles.

Objective: This study investigated the metabolic status of the spent culture media from embryos of patients with repeated implantation failure (RIF) undergoing in vitro fertilization-intracytoplasmic sperm injection cycles in comparison with the embryos from healthy fertile women. Methods: Metabolite levels in spent culture media were assessed and compared between embryos from RIF patients (n=35) and oocyte donors as controls (n=15). Protein levels of insulin-like growth factor 1 (IGF-1) were determined using Western blotting. Concentrations of glucose, pyruvate, and lactate were measured using spectrophotometry. Ionic colorimetric assay kits were utilized to analyze the concentrations of sodium, chloride, calcium, and magnesium ions. High-performance liquid chromatography was employed to measure the concentrations of glutamic acid, aspartic acid, methionine, phenylalanine, and histidine. Results: Glucose consumption and lactate secretion were higher in the control group than in the RIF group. The magnesium concentration was significantly higher in the control group than in the RIF group, but glutamic acid and aspartic acid concentrations were lower in the control group than in the RIF patients (p<0.05). The levels of IGF-1, sodium, calcium, chloride, methionine, histidine, and phenylalanine did not show statistically significant differences between the two groups. Conclusion: The metabolic profile of the culture medium of the embryos in the RIF group differed from that of the control group. These findings suggest potential factors that may affect implantation capacity in RIF patients and provide a new perspective on embryo selection.

Implication of Novel BMP15 and GDF9 Variants in Unexpected Poor Ovarian Response.

Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.

Endometrial scratching in unexplained repeated implantation failure causes two competing forces, angiogenesis and anti-angiogenesis: An RCT study.

Background: A significant association between endometrial vascularity and pregnancy has been shown in previous research, while poor vascularization was attributed to repeated implantation failure (RIF). One possible approach to enhance angiogenesis for successful implantation is endometrial scratching (ES). Objective: The purpose was to investigate endometrial responses to scratching by profiling angiogenesis-related gene expression in unexplained RIF participants. Materials and Methods: In this randomized controlled trial study, 20 infertile women with unexplained RIF were assigned to 2 groups by the balanced block randomization method (n = 10/each group): the intervention group (group A) (who received ES in the follicular phase) and the control group (group B). Endometrial biopsy was performed in the secretory phase. Gene expression profiling was performed using a polymerase chain reaction-array kit for human-angiogenic growth factors. The implantation and clinical pregnancy rates were also assessed. Results: Among the angiogenesis-promoting genes, FGF1, FGF13, FGF2, TGFA, ANG, ANGPT1, and VEGFA were significantly upregulated (p < 0.05). IL12A (an angiogenesis-inhibiting cytokine) was significantly upregulated (p < 0.01). In contrast, 15 genes with angiogenesis-related functions, including CXCL11, CXCL13, CXCL3, CXCL5, CXCL6, EREG, FIGF, FST, IL10, LEP, PPBP, PROK1, RHOB, TNF, and TYMP, were downregulated after ES. No significant differences were observed between the intervention (group A) and control (group B) groups in terms of implantation (43.75% vs. 28.57%) or clinical pregnancy rates (75% vs. 57.1%). Conclusion: ES induced significant alterations in the expression of angiogenesis-related genes, with notable up/downregulation of key angiogenic/antiangiogenic factors. These findings enhance our understanding of the molecular responses triggered by ES, underscoring the potential influence of ES on the complex processes of angiogenesis crucial for implantation.

In vitro effects of plasma rich in growth factors on human teratozoospermic semen samples.

There is a correlation between teratozoospermia and production of reactive oxygen species leading to poor assisted reproductive techniques outcomes. This study aimed to examine the effect of plasma-rich in growth factors (PRGF) on teratozoospermic samples. Twenty-five teratozoospermic samples were included in this study. After sperm preparation, it was divided into four groups, including 0 (control), 1, 5, and 10% PRGF. Sperm motility, viability (eosin-nigrosin staining), morphology (Papanicolaou staining), DNA fragmentation (sperm chromatin dispersion test), mitochondrial membrane potential (JC-1 staining by flow cytometry), and lipid peroxidation (measurement of malondialdehyde, MDA) were evaluated before and after 1 h of incubation with or without PRGF. Our results showed that after 1 h of incubation, the addition of 1% PRGF improved sperm progressive motility (47.72 ± 13.76%) compared to the control group (17.36 ± 8.50%) (p < 0.001). Also, 1% PRGF preserved the sperm's total motility (77.50 ± 13.28% vs. 65.63 ± 19.03%, for 1% PRGF and control, respectively) and viability after incubation. The rate of normal sperm morphology was the same between different groups. Higher mitochondrial membrane potential and lower DNA fragmentation were also observed in sperm treated with different concentrations of PRGF compared to the control group, but the differences were non-significant. The MDA levels were significantly decreased in PRGF-treated groups compared to the control group (0.99 ± 0.62, 0.95 ± 0.33, 0.95 ± 0.79, and 1.49 ± 0.27 for 1% PRGF, 5% PRGF, 10% PRGF and control, respectively). Based on our results, it seems that PRGF incubation can improve sperm parameters and especially decrease the level of malondialdehyde as an indicator of oxidative stress, which is one of the main problems of teratozoospermic samples.

TLR-1, TLR-2, and TLR-6 MYD88-dependent signaling pathway: A potential factor in the interaction of high-DNA fragmentation human sperm with fallopian tube epithelial cells.

OBJECTIVE: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. METHODS: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. RESULTS: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1ß, IL-6, IL-12, interferon α (IFN-α), IFN-ß, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. CONCLUSION: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

Expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparin-binding epidermal growth factor-like growth factor, and fibroblast growth factor 2 genes in the female reproductive tracts of women with ectopic pregnancy: A case-control study.

Background: Ectopic pregnancy (EP) is defined as embryo implantation in a location other than the uterine cavity. Objective: We aimed to evaluate the expression of several genes, which may play a role in EP, in the ampulla region of fallopian tubes and endometrial tissue of women with EP. Materials and Methods: In this case-control study, 5 women who underwent salpingectomy due to EP, comprised the 5 pseudo-pregnant women as a control group. These participants referred to the Royan Institute, Shariati, and Arash hospital, Tehran, Iran during 2019-2021. We evaluated the expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparin-binding epidermal growth factor-like growth factor (HBEGF), and fibroblast growth factor 2 genes in the fallopian tube and endometrium of EP cases by real-time polymerase chain reaction using specific primers. Results: The vascular endothelial growth factor expression was significantly higher in the ampulla region of the controls. However, no significant differences were observed in endometrial tissue. Assessments of colony-stimulating factor-1 and fibroblast growth factor 2 showed no significant differences between the case and control groups. HBEGF showed significantly higher expression in the ampulla region of EP cases, but no significant difference was observed in HBEGF expression in the endometrial tissues of the study groups. Mucin-1expression was significantly higher in both study regions of the EP cases. Conclusion: Our results have strongly suggested that these genes play important roles in proper implantation, and disruptions in their expression patterns could lead to EP. However, more studies are needed to confirm the current findings.