Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility.

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Up-regulated mRNA expression of VEGFA receptors (FLT1 and KDR) in placentas after assisted reproductive technology fertilization.

Placental angiogenesis is a pivotal process for feto-maternal circulation and ensures efficient development of the placenta throughout pregnancy. Many factors during in vitro fertilization and embryo transfer procedures may affect placental gene expression and fetus development. The present study aimed to identify differences in angiogenesis-related gene (VEGFA, FGF2, FLT1, and KDR) expression profiles in placentas after assisted reproductive technology fertilization and natural conception in healthy women. In a case-control study, term placentas were collected from Caucasian women after assisted reproductive technology fertilization (N = 20) and after natural conception in women with uncomplicated pregnancy (N = 9). The mRNA expression in placentas was examined for VEGFA, FGF2, FLT1, and KDR genes by real-time quantitative polymerase chain reaction (RT-qPCR). Group stratification was performed for comparison of investigated genes between the type of embryo transferred (fresh/frozen), place of tissue donation (center/margin), and newborns' gender (male/female). In the ART placentas, significant down-regulation of VEGFA gene (p = 0.016) and up-regulation of FLT1 (p = 0.026) and KDR (p < 0.001) gene receptors were observed. Genes encoding VEGFA receptors were up-regulated in both fresh (ET) and frozen (FET) embryo transfer groups compared to controls. For the FLT1 gene, a statistically significant difference was observed between the frozen embryo transfer group and the controls (p = 0.032). Relative expression of KDR was significantly higher for both embryo transfer groups compared to controls (p < 0.001) and between ET and FET (p = 0.002). No statistically significant differences were observed between placental expression in different places of tissue donation and newborns' gender. We observed differences in the placental expression of VEGFA and its receptors FLT1 and KDR in pregnancies after assisted reproductive technology compared to naturally conceived pregnancies. More research is needed to clarify these alterations that may affect placental development and fetal health.

ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia.

Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40-50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells-TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.

Polymorphic Variants of Genes Encoding Angiogenesis-Related Factors in Infertile Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a global health issue affecting a significant number of infertile women who undergo in vitro fertilization (IVF) cycles. Extensive vasculogenesis and angiogenesis occur in both maternal and fetal placental tissues, and vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family molecules and their receptors are potent angiogenic mediators in the placenta. Five single nucleotide polymorphisms (SNPs) in the genes encoding angiogenesis-related factors were selected and genotyped in 247 women who had undergone the ART procedure and 120 healthy controls. Genotyping was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A variant of the kinase insertion domain receptor (KDR) gene (rs2071559) was associated with an increased risk of infertility after adjusting for age and BMI (OR = 0.64; 95% CI: 0.45-0.91, p = 0.013 in a log-additive model). Vascular endothelial growth factor A (VEGFA) rs699947 was associated with an increased risk of recurrent implantation failures under a dominant (OR = 2.34; 95% CI: 1.11-4.94, padj. = 0.022) and a log-additive model (OR = 0.65; 95% CI 0.43-0.99, padj. = 0.038). Variants of the KDR gene (rs1870377, rs2071559) in the whole group were in linkage equilibrium (D' = 0.25, r2 = 0.025). Gene-gene interaction analysis showed the strongest interactions between the KDR gene SNPs rs2071559-rs1870377 (p = 0.004) and KDR rs1870377-VEGFA rs699947 (p = 0.030). Our study revealed that the KDR gene rs2071559 variant may be associated with infertility and rs699947 VEGFA with an increased risk of recurrent implantation failures in infertile ART treated Polish women.

Expression of estrogen receptors, PELP1, and SRC in human spermatozoa and their associations with semen quality.

Sperm cells are target cells for both estrogens and xenoestrogens. Due to the specific structure of spermatozoa, these hormonal compounds may act on sperm in a non-genomic mechanism only. However, the ESR-mediated signaling pathways are still poorly understood. In this study, we obtained 119 samples from male participants of Caucasian descent who donated semen for standard analysis. We analyzed gene expression of estrogen receptors (ESR1 and ESR2) and their coregulators-proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and cellular kinase c-Src (SRC). RNA level was established using reverse-transcribed RNA as a template, followed by a polymerase chain reaction. Proteins' presence was confirmed by western blot and immunocytochemistry techniques. "Normal" values of semen parameters were defined as follows: > 32% sperm with progressive motility, > 4% sperm cells with normal morphology, > 15 × 106 sperm per mL, > 58% live spermatozoa and leukocyte amount < 106 cells per mL, according to WHO 2010 reference. Semen parameters that deviated from these "normal" values were labeled as "abnormal". Gene expression ratios revealed significant, moderate, and negative correlations for ESR1/ESR2 and weak, negative ESR2/PELP1 correlations in the subgroup of patients with abnormal values of semen parameters. In addition, SRC/PELP1 was moderately and positively correlated in the subgroup with parameters within the reference values established by WHO 2010. Our study showed that both PELP1 scaffolding protein and SRC kinase might influence semen quality via ESRs. It seems that not the expression of a single gene may affect the sperm quality, but more gene-to-gene mutual ratio. Characterization of estrogen-signaling pathway-related genes' modulated expression in sperm cells could aid in better understanding sperm biology and quality.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia.

BACKGROUND: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. OBJECTIVE: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). MATERIALS AND METHODS: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. RESULTS: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. DISCUSSION AND CONCLUSION: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.

Effects of TIMP1 rs4898 Gene Polymorphism on Early-Onset Preeclampsia Development and Placenta Weight.

Introduction: Some evidence indicates that the improper trophoblast invasion of maternal spiral arteries could be caused by an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to preeclampsia (PE) development. This study aimed to assess the potential role of MMP1, MMP9, TIMP1 and TIMP2 gene polymorphisms in the pathogenesis of PE. Materials and methods: A total of 308 Polish women, 115 preeclamptic (55 with early-onset preeclampsia [EOPE], 60 with late-onset preeclampsia [LOPE]) and 193 healthy pregnant women, all of Caucasian origin, were recruited to the study. PE was diagnosed following the ACOG criteria. The polymorphic variants of the MMP-TIMP pathway (MMP1 rs1799750, MMP9 rs17576, MMP9 rs17577, TIMP1 rs4898, TIMP2 rs2277698, TIMP2 rs55743137) were genotyped by polymerase chain reaction and restriction fragment length polymorphism. Results: Analyzing all SNPs in the MMP-TIMP pathway, no significant differences in allele frequencies between preeclamptic women and controls were observed. However, comparing the EOPE and LOPE groups with each other, we observed a statistically significant difference between them for the TIMP1 rs4898 variant. In the whole group of 308 women, the mean placenta weight was the lowest in carriers of the rs4898 CC genotype. Post hoc analysis revealed significant differences between CC-CT (p = 0.0209) and CC-TT (p = 0.0469). Additionally, during allele analysis, a statistically significant difference in the mean placenta weight (for C allele 529.32 ± 157.11 g, for T allele 560.24 ± 162.24 g, p = 0.021) was also observed. Conclusion: Our findings suggest a relationship between TIMP1 rs4898 (372T > C) polymorphism and increased risk of early-onset preeclampsia in a population of pregnant Polish women. Our data suggest that the TIMP1 rs4898 C allele might be associated with increased risk for early-onset, but not for late-onset preeclampsia. To evaluate the role of the TIMP1 polymorphic variants in the etiopathology of preeclampsia, further studies with a larger sample size are needed.

Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males.

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.

Intensified Hyposensitization Is an Effective Treatment of Postorgasmic Illness Syndrome (POIS).

INTRODUCTION: Postorgasmic illness syndrome (POIS) is an extremely rare urogenital disease which significantly and negatively impacts the functioning and sexual activity. AIMS: A 34-year-old male presented with POIS symptoms and confirmed the allergic component of the POIS. Intensified immunotherapy with autologous semen was recommended. METHODS: The treatment lasted 14 months and included 20 visits. Modified and intensified subcutaneous immunotherapy in both forearms significantly shortened the therapy and improved the outcome, with high-tolerance and no adverse effects or hyperactive responses. MAIN OUTCOME MEASURE: Improvement in POIS symptoms through the use of intensified immunotherapy with autologous semen. RESULTS: The improvement was significant enough to allow for higher sexual activity, and gradual resumption of private and professional activity. CONCLUSION: Intensified immunotherapy with autologous semen seems an effective and safe option for treating patients with suspected immune-allergenic POIS. To the best of our knowledge, this has been the first such intensive and effective allergen-specific immunotherapy of POIS. Wrotynska-Barczynska J, Swat. Swat E, Berger A, et al. Intensified Hyposensitization Is an Effective Treatment of Postorgasmic Illness Syndrome (POIS). Sex Med 2022;10:100474.

Relationship of Postoperative Pain and PONV after Minimally Invasive Surgery with the Serotonin Concentrations and Receptors' Gene Polymorphisms.

(1) Background: there is a steady increase in the number of procedures performed via minimally invasive surgery, which have many benefits, but post-operative nausea and vomiting (PONV) and significant pain are still a common problem (2) Methods: 300 infertile women (18-40 years old) undergoing minimal invasive surgery. Interventions: laparoscopy and hysteroscopy performing, evaluation of postoperative symptoms, serotonin concentrations assessment, identify genetic polymorphisms. (3) Results: serotonin concentrations were significantly lower among women who required opioids (p = 0.006). The presence of the GG genotype in the rs6318 polymorphism of the 5HTR2C gene had a protective effect on PONV (OR = 0.503; C.I. = [0.300-0.841]; p = 0.008), when the GG variant of the rs11214763 polymorphism of the 5HTR3B gene, when the risk of PONV was 1.65-fold higher (OR = 1.652; C.I. = [1.003-2.723]; p = 0.048). Pain intensity was significantly higher among women with GG genotype of the rs6296 polymorphism of the 5HTR1B gene (OR = 1.660; C.I. = [1.052-2.622]; p = 0.029).; (4) Conclusions: the evaluation of serotonin concentration predicts requirement for opioid pain relief medication. The polymorphisms of the serotonin receptors affect the intensity of postoperative complaints.

Biomolecular Markers of Recurrent Implantation Failure-A Review.

Currently, infertility affects 8-12% of reproductive age couples worldwide, a problem that also affects women suffering from recurrent implantation failure (RIF). RIF is a complex condition resulting from many physiological and molecular mechanisms involving dynamic endometrium-blastocyst interaction. The most important are the endometrial receptivity process, decidualization, trophoblast invasion, and blastocyst nesting. Although the exact multifactorial pathogenesis of RIF remains unclear, many studies have suggested the association between hormone level imbalance, disturbances of angiogenic and immunomodulatory factors, certain genetic polymorphisms, and occurrence of RIF. These studies were performed in quite small groups. Additionally, the results are inconsistent between ethnicities. The present review briefly summarizes the importance of factors involved in RIF development that could also serve as diagnostic determinants. Moreover, our review could constitute part of a new platform for discovery of novel diagnostic and therapeutic solutions for RIF.

Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics.

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

Increased expression of PELP1 in human sperm is correlated with decreased semen quality.

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein involved in both genomic and nongenomic estrogen signal transduction pathways. To date, the role of PELP1 protein has yet to be characterized in human sperm and has not been associated with sperm parameters. To confirm the presence of PELP1 in human sperm, fresh semen samples were obtained from 178 donors. The study was designed to establish both mRNA and protein presence, and protein cellular localization. Additionally, the number of PELP1-positive spermatozoa was analyzed in men with normal and abnormal semen parameters. Sperm parameters were assessed according to the World Health Organization (WHO) 2010 standards. The presence of PELP1 in spermatozoa was investigated using four precise, independent techniques. The qualitative presence of transcripts and protein was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and western blot protocols, respectively. The cellular localization of PELP1 was investigated by immunocytochemistry. Quantitative analysis of PELP1-positive cells was done by flow cytometry. PELP1 mRNA and protein was confirmed in spermatozoa. Immunocytochemical analysis identified the presence of PELP1 in the midpieces of human sperm irrespective of sperm parameters. Becton Dickinson fluorescence-activated cell sorting (FACSCalibur™) analysis revealed a significantly lower number of PELP1-positive cells in males with normal semen parameters versus abnormal samples (42.78% ± 11.77% vs 61.05% ± 21.70%, respectively; P = 0.014). The assessment of PELP1 may be a time-saving method used to obtain information about sperm quality. The results of our study suggest that PEPL1 may be utilized as an indicator of sperm quality; thereby, PELP1 may be an additional biomarker useful in the evaluation of male infertility.

Planning and preparation for pregnancy among women with and without a history of infertility.

OBJECTIVES: Preconception counseling, maternal health-related habits, diet, folic acid consumption, substances abuse, may all impact the outcome of pregnancy. The aim of this study was to compare the planning and preparation for pregnancy among pregnant women with and without infertility. MATERIAL AND METHODS: A survey of health behaviors prior to and during pregnancy that could affect pregnancy outcomes, including laboratory tests performed, stimulant usage, initiation of prenatal care, and folic acid intake, was conducted among 400 pregnant women. The study group included 121 women (30.25%) diagnosed with prior infertility, while the control group included 279 women (69.74%) who did not report any problems conceiving. RESULTS: All patients (100%) from the study group and 70,97% from the control group planned their pregnancy(p < 0.0001). Patients in the study group performed significantly more laboratory tests prior to pregnancy, including: complete blood count, urine analysis, fasting blood glucose concentration, testing for toxoplasmosis, and Pap smear, compared with the control group (p < 0.0001). There was no difference between groups regarding the knowledge of when and why folic acid supplementation is required (p > 0.05). CONCLUSIONS: Effective education of women, regarding pregnancy planning and behaviours, that may impact pregnancy outcome is still a serious challange to public health in Poland. Our study indicates that reaching general population with the education is most important to achieve best results in preconceptional care.

Manual vs. computer-assisted sperm analysis: can CASA replace manual assessment of human semen in clinical practice?

OBJECTIVES: The aim of the study was to check the quality of computer-assisted sperm analysis (CASA) system in comparison to the reference manual method as well as standardization of the computer-assisted semen assessment. MATERIAL AND METHODS: The study was conducted between January and June 2015 at the Andrology Laboratory of the Division of Infertility and Reproductive Endocrinology, Poznan University of Medical Sciences, Poland. The study group consisted of 230 men who gave sperm samples for the first time in our center as part of an infertility investigation. The samples underwent manual and computer-assisted assessment of concentration, motility and morphology. A total of 184 samples were examined twice: manually, according to the 2010 WHO recommendations, and with CASA, using the program set-tings provided by the manufacturer. Additionally, 46 samples underwent two manual analyses and two computer-assisted analyses. The p-value of p < 0.05 was considered as statistically significant. RESULTS: Statistically significant differences were found between all of the investigated sperm parameters, except for non-progressive motility, measured with CASA and manually. In the group of patients where all analyses with each method were performed twice on the same sample we found no significant differences between both assessments of the same probe, neither in the samples analyzed manually nor with CASA, although standard deviation was higher in the CASA group. CONCLUSIONS: Our results suggest that computer-assisted sperm analysis requires further improvement for a wider application in clinical practice.

The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males.

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract.

17ß-estradiol modifies human spermatozoa mitochondrial function in vitro.

BACKGROUND: It is assumed that spermatozoa are target cells for estrogens however, the mechanism of their action is not fully understood. The aim of this study was to investigate the influence of 17ß-estradiol (E2) on the human spermatozoa mitochondrial function. METHODS: The effects on spermatozoa of E2 at final concentrations of 10(-10), 10(-8) and 10(-6) M were studied regarding the following phenomena: (1) kinetics of intracellular free calcium ions changes (using Fluo-3), (2) mitochondrial membrane potential ΔΨm (using JC-1 fluorochrome), (3) production of superoxide anion in mitochondria (using MitoSOX RED dye), (4) spermatozoa vitality (propidium iodide staining) and (5) phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein). RESULTS: E2 initiated rapid (within a few seconds) dose dependent increase of intracellular free calcium ions concentration. E2 was changing the mitochondrial membrane potential: 10(-8) M initiated significant increase of percentage of high ΔΨm spermatozoa while the 10(-6) M induced significant decrease of high ΔΨm cells. In spermatozoa stimulated with E2 10(-6) M a significant increase of mitochondrial superoxide anion level was observed. 2 h incubation of spermatozoa with E2 did not alter cells vitality nor stimulated phosphatidylserine membrane translocation, for all three doses. CONCLUSIONS: 17ß-estradiol affected the human spermatozoa mitochondrial function. E2 in low concentration improved while in high concentration might deteriorate mitochondrial function.

17ß-estradiol and xenoestrogens reveal synergistic effect on mitochondria of human sperm.

OBJECTIVES: The aim of the study was to investigate the influence of 17ß-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human sper-matozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. MATERIAL AND METHODS: The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye). RESULTS: Two-hour incubation of spermatozoa with 17ß-estradiol, genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17ß-estradiol or bisphenol-A sepa-rately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. CONCLUSIONS: It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17ß-estradiol and xenoestrogens. The reaction to the 17ß-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17ß-estradiol.

Effect of growth differentiation factor­9 C447T and G546A polymorphisms on the outcomes of in vitro fertilization.

Single nucleotide polymorphisms (SNPs) in the growth differentiation factor (GDF)­9 gene are associated with premature ovarian failure, insufficient ovarian stimulation and a poor in vitro fertilization (IVF) score in women with diminished ovarian reserve. The aim of the present study was to assess the effect of C447T (rs254286) and G546A (rs10491279) SNPs on ovary stimulation response, oocyte quality, fertilization rate and outcome of clinical pregnancy in an infertile population of Polish females (n=86) treated with IVF. The present study also included a group of fertile women (n=202). The P­trend value, calculated for the GDF­9 C447T transition in infertile women, was statistically significant and were equal to 0.0195. A significant association of the GDF­9 C447T SNP was observed with infertility for the C/C vs. T/T + C/T model (OR= 2.140; 95% CI=1.043­4.393; P=0.0349). The GDF­9 G546A SNP was significantly associated with the G/A vs. G/G model with poor ovarian stimulation (OR=9.303; 95% CI=2.568­33.745; P=0.0008) and poor fertilization rate (OR=2.981; 95% CI=1.033­8.607; P=0.0385). For the GDF­9 C447T SNP, a significant association was observed between the C/C + C/T vs. T/T model and a poor ovarian stimulation response (OR=15.309; 95% CI=0.875­267.83; P=0.0078), and a poor fertilization rate (OR=4.842; 95% CI=1.310­17.901; P=0.0121). The present genetic evaluation revealed associations between IVF outcomes and the GDF­9 A546G and C447T SNPs. Additionally, these results indicated that the GDF­9 C447T SNP is a possible candidate genetic risk factor for female infertility in the Polish population.