Genetic Insights into Azoospermia and Severe Oligozoospermia: Discovering Seven SNPs through GWAS and In Silico Analysis.

Azoospermia and severe oligozoospermia represent the most extreme forms of male infertility. Despite their prevalence, the genetic foundations of these conditions are not well understood, with only a limited number of genetic factors identified so far. This study aimed to identify single-nucleotide polymorphisms (SNPs) linked to both azoospermia and severe oligozoospermia. We conducted a genome-wide association study (GWAS) involving 280 Greek males with normal semen parameters and 85 Greek males diagnosed with either azoospermia or severe oligozoospermia. Following rigorous quality control measures, our analysis identified seven SNPs associated with azoospermia/severe oligozoospermia. An in silico functional annotation was subsequently used to further investigate their role. These SNPs, found in regions not previously associated with male reproductive disorders, suggest novel genetic pathways that may contribute to these forms of infertility and pave the way for future studies. Additionally, this study sheds light on the significant role of noncoding RNAs in the pathogenesis of male infertility, with three of the identified SNPs situated in long intergenic non-coding RNAs (lincRNAs). Our findings highlight the intricate genetic landscape of azoospermia and severe oligozoospermia, underlining the necessity for more detailed studies to fully grasp the underlying mechanisms and their potential for informing diagnostic and therapeutic strategies.

Elucidating the Role of OXPHOS Variants in Asthenozoospermia: Insights from Whole Genome Sequencing and an In Silico Analysis.

Infertility is a global health challenge that affects an estimated 72.4 million people worldwide. Between 30 and 50% of these cases involve male factors, showcasing the complex nature of male infertility, which can be attributed to both environmental and genetic determinants. Asthenozoospermia, a condition characterized by reduced sperm motility, stands out as a significant contributor to male infertility. This study explores the involvement of the mitochondrial oxidative phosphorylation (OXPHOS) system, crucial for ATP production and sperm motility, in asthenozoospermia. Through whole-genome sequencing and in silico analysis, our aim was to identify and characterize OXPHOS gene variants specific to individuals with asthenozoospermia. Our analysis identified 680,099 unique variants, with 309 located within OXPHOS genes. Nine of these variants were prioritized due to their significant implications, such as potential associations with diseases, effects on gene expression, protein function, etc. Interestingly, none of these variants had been previously associated with male infertility, opening up new avenues for research. Thus, through our comprehensive approach, we provide valuable insights into the genetic factors that influence sperm motility, laying the foundation for future research in the field of male infertility.

Unveiling the Genetic Complexity of Teratozoospermia: Integrated Genomic Analysis Reveals Novel Insights into lncRNAs' Role in Male Infertility.

Male infertility is a global health issue, affecting over 20 million men worldwide. Genetic factors are crucial in various male infertility forms, including teratozoospermia. Nonetheless, the genetic causes of male infertility remain largely unexplored. In this study, we employed whole-genome sequencing and RNA expression analysis to detect differentially expressed (DE) long-noncoding RNAs (lncRNAs) in teratozoospermia, along with mutations that are exclusive to teratozoospermic individuals within these DE lncRNAs regions. Bioinformatic tools were used to assess variants' impact on lncRNA structure, function, and lncRNA-miRNA interactions. Our analysis identified 1166 unique mutations in teratozoospermic men within DE lncRNAs, distinguishing them from normozoospermic men. Among these, 64 variants in 23 lncRNAs showed potential regulatory roles, 7 variants affected 4 lncRNA structures, while 37 variants in 17 lncRNAs caused miRNA target loss or gain. Pathway Enrichment and Gene Ontology analyses of the genes targeted by the affected miRNAs revealed dysregulated pathways in teratozoospermia and a link between male infertility and cancer. This study lists novel variants and lncRNAs associated for the first time with teratozoospermia. These findings pave the way for future studies aiming to enhance diagnosis and therapy in the field of male infertility.

Replicating a GWAS: two novel candidate markers for oligospermia in Greek population.

Genome-wide association studies have paved the way for the discovery of new markers regarding many diseases, including male infertility. A previous study on Caucasians highlighted 172 polymorphisms for their putative association with male infertility and we attempted to replicate these findings on our dataset comprising of Greek male individuals (n = 360). We retrieved 59 out of 172 polymorphisms and tested for all association models on 278 normospermic men and 82 patients with an abnormal seminogram, later separated into oligozoospermic and asthenozoospermic groups. Our findings indicate that two SNPs (rs2296225 in KIF17, rs7224496 in SMYD4) are associated with male infertility in the Greek population and have not been recorded in literature as of yet. These novel markers need further validation via additional studies and an increased individual number. All in all, replication studies, possess the power to validate existing polymorphisms found across all population and thus increase both statistical significance as well as identify novel potentially diagnostic markers.

Embryonic Stem Cells in Clinical Trials: Current Overview of Developments and Challenges.

The first isolation of human embryonic stem cells (hESC) reported in the late 90s opened a new window to promising possibilities in the fields of human developmental biology and regenerative medicine. Subsequently, the differentiation of hESC lines into different precursor cells showed their potential in treating different incurable diseases. However, this promising field has consistently had remarkable ethical and experimental limitations. This paper is a review of clinical trial studies dealing with hESC and their advantages, limitations, and other specific concerns. Some of the hESC limitations have been solved, and several clinical trial studies are ongoing so that recent clinical trials have strived to improve the clinical applications of hESC, especially in macular degeneration and neurodegenerative diseases. However, regarding hESC-based therapy, several important issues need more research and discussion. Despite considerable studies to Date, hESC-based therapy is not available for conventional clinical applications, and more studies and data are needed to overcome current clinical and ethical limitations. When all the limitations of Embryonic stem cells (ESC) are wholly resolved, perhaps hESC can become superior to the existing stem cell sources. This overview will be beneficial for understanding the standard and promising applications of cell and tissue-based therapeutic approaches and for developing novel therapeutic applications of hESC.

The impact of fresh and frozen testicular tissue quality on embryological and clinical outcomes.

Our ability to predict the potential of testicular spermatozoa to support embryonic development is still limited. Although motility of testicular spermatozoa is associated with embryo development, the impact of morphology and the presence of spermatozoa in the testicular sample has not been previously researched. Moreover, while the majority of data indicate no effect of cryopreservation, there are studies reporting impaired clinical outcomes due to testicular cryopreservation. In a retrospective study, 118 ICSI-TESE cycles were analysed to study the impact of (a) total quality of testicular tissue, (b) testicular tissue cryopreservation and (c) presence/motility/morphology of testicular spermatozoa in fertilisation rate, embryonic development, clinical pregnancy (CPR), ongoing pregnancy (OPR) and live birth rate (LBR). Results showed that fertilisation rate was significantly affected by both total quality of testicular tissue (p < .05) and rare presence of spermatozoa (p < .01). Moreover, total tissue quality (p < .01), cryopreservation of low-quality samples (p < .01), absence of motile testicular spermatozoa (p < .01) and poor spermatozoa morphology (p < .05) had a negative impact on the number of good quality day 3 embryos. CPR, OPR or LBR was not affected by any parameters examined. Our data suggest that the quality of testicular tissue influences both fertilisation rate and embryo development. Moreover, cryopreservation of low-quality testicular samples has a negative impact on the number of available embryos for transfer.

Genome-wide association study identifies candidate markers related to lincRNAs associated with male infertility in the Greek population.

BACKGROUND: Male infertility is currently one of the most common problems faced by couples worldwide. We performed a GWAS on Greek population and gathered statistically significant SNPs in order to investigate whether they lie within or near lncRNA regions. OBJECTIVES: The aim of this study was to investigate whether polymorphisms on or near lncRNAs affect interactions with miRNAs and can cause male infertility. MATERIALS AND METHODS: In the present study, a GWAS was conducted, using samples from 159 individuals (83 normozoospermic individuals and 76 patients of known fertility issues). Standard procedures for quality controls and association testing were followed, based on case-control testing. RESULTS: We detected six lncRNAs (LINC02231, LINC00347, LINC02134, NCRNA00157, LINC02493, Lnc-CASK-1) that are associated with male infertility through their interaction with miRNAs. Furthermore, we identified the genes targeted by those miRNAs and highlighted their functions in spermatogenesis and the fertilization process. DISCUSSION AND CONCLUSION: lncRNAs are involved in spermatogenesis through their interaction with miRNAs. Thus, their study is very important, and it may contribute to the understanding of the molecular mechanisms underlying male infertility.

Embryos from vitrified vs. fresh oocytes in an oocyte donation program: a comparative morphokinetic analysis.

OBJECTIVE: To compare the morphokinetic patterns of human embryos originating from vitrified oocytes (VITRI group) with those derived from freshly collected oocytes (CONTROL group) in oocyte donation cycles. DESIGN: This is a retrospective observational study. SETTING: Embryolab Fertility Clinic, Embryology Lab, Thessaloniki, Greece. PATIENT(S): The study included embryos from 421 vitrified oocytes from 58 oocyte donation cycles and 196 fresh oocytes from 23 oocyte donation cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Key time parameters, dynamic events, fertilization rates, degeneration rates, cleavage rates, blastocyst rates, pregnancy rates, clinical pregnancy rates, implantation rates, and live birth rates were estimated. RESULTS: The mean survival rate of vitrified oocytes was 92.58% (±7.42%). Fertilization rates were significantly different between the 2 groups (VITRI group: 71.92% ± 20.29% and CONTROL group: 80.65% ± 15.22%) whereas the degeneration, cleavage, blastocyst, pregnancy, clinical pregnancy, ongoing pregnancy, implantation, and live birth rates were not significantly different between embryos derived from fresh or vitrified oocytes. Time-lapse analysis showed no significant difference in any key time parameter. However, when examining dynamic parameters, first cell cycle (CC1) (t2 - tPB2: from the second polar body extrusion (tPB2) up to 2 cells (t2)) showed a significant difference whereas CC1a (t2 - tPNf: from fading of the pronuclei (tPNf) up to 2 cells (t2)) was at the threshold of significance. CONCLUSION(S): CC1 in vitrified oocytes exhibited a comparatively slower progression in contrast to fresh oocytes. Conversely, CC1a in vitrified oocytes demonstrated faster progression compared with fresh oocytes. It is worth noting that these temporary deviations had minimal impact on the subsequent development. Despite the clinical outcomes showing a decrease in the vitrified group, none of them reached statistical significance. This lack of significance could be attributed to the limited sample size of the study.

Double vitrification of embryos adversely affects clinical outcomes.

OBJECTIVE: To evaluate the impact of double embryo vitrification on clinical outcomes. METHODS: This retrospective cohort study included data from January 2013 to March 2021. The study group included women aged 33.3±5.7 years with double-vitrified embryos (n=381), while the control group included women aged 32.1±6.7 years with embryos vitrified once (n=780), all transferred at the blastocyst stage. The primary endpoint was live birth rate (LBR), and secondary endpoints included percent positive ßHCG test, clinical/ongoing pregnancy rates, miscarriage/biochemical pregnancy rates and birthweight. RESULTS: LBR was significantly lower in double-vitrified embryos (30.2%) than in embryos vitrified once (45.6%, p<.05). Similarly, double-vitrified embryos were associated with significantly lower positive ßHCG tests (46% vs. 63.3%, p<.05) and clinical (34.9% vs. 52.2%, p<.05) and ongoing pregnancy (31.3% vs. 47.3%, p<.05) rates compared to embryos vitrified once. However, biochemical pregnancy (double vitrified: 24.1% vs. vitrified once: 17.9%, p>.05) and miscarriage rates (double vitrified: 10.2% vs. vitrified once: 9.4%, p>.05), as well as mean birthweight (double-vitrified embryos: 2950g vs. embryos vitrified once: 2837g, p>.05) did not differ significantly between two groups. On a secondary comparison, amongst double-vitrified embryos, the subgroup that was cultured for more than 24 hours between warming and second vitrification achieved significantly higher positive ßHCG tests (49%) and clinical pregnancy (38%) rates, compared to embryos re-vitrified on the same day of warming (31.8% and 20.5%, respectively, p<.05). Nevertheless, LBR did not differ significantly amongst these study-group embryos (embryos that remained in culture for more than 24 hours: 32.2% vs. embryos that were re-vitrified on warming day: 20.5%, p>.05). CONCLUSIONS: Double vitrification of embryos adversely affects clinical outcomes. However, it represents a valuable option concerning embryo wastage, with acceptable success rates.

Gene-by-Sex Interactions: Genome-Wide Association Study Reveals Five SNPs Associated with Obesity and Overweight in a Male Population.

Obesity is a chronic health problem associated with severe complications and with an increasing prevalence in the Western world. Body-fat composition and distribution are closely associated with obesity, but the human body's composition is a sexually dimorphic trait, as differences between the two sexes are evident even from fetal life. The effect of sex hormones contributes to this phenomenon. However, studies investigating gene-by-sex interactions for obesity are limited. Therefore, the aim of the present study was to identify single-nucleotide polymorphisms (SNPs) associated with obesity and overweight in a male population. A genome-wide association study (GWAS) that included 104 control, 125 overweight, and 61 obese subjects revealed four SNPs associated with overweight (rs7818910, rs7863750, rs1554116, and rs7500401) and one SNP (rs114252547) associated with obesity in males. An in silico functional annotation was subsequently used to further investigate their role. Most of the SNPs were found in genes regulating energy metabolism and homeostasis, and some of them were expression quantitative trait loci (eQTL). These findings contribute to the understanding of the molecular mechanisms underlying obesity-related traits, especially in males, and pave the road for future research toward the improvement of the diagnosis and therapy of obese individuals.

Whole-Genome Profile of Greek Patients with Teratozοοspermia: Identification of Candidate Variants and Genes.

Male infertility is a global health problem that affects a large number of couples worldwide. It can be categorized into specific subtypes, including teratozoospermia. The present study aimed to identify new variants associated with teratozoospermia in the Greek population and to explore the role of genes on which these were identified. For this reason, whole-genome sequencing (WGS) was performed on normozoospermic and teratozoospermic individuals, and after selecting only variants found in teratozoospermic men, these were further prioritized using a wide range of tools, functional and predictive algorithms, etc. An average of 600,000 variants were identified, and of them, 61 were characterized as high impact and 153 as moderate impact. Many of these are mapped in genes previously associated with male infertility, yet others are related for the first time to teratozoospermia. Furthermore, pathway enrichment analysis and Gene ontology (GO) analyses revealed the important role of the extracellular matrix in teratozoospermia. Therefore, the present study confirms the contribution of genes studied in the past to male infertility and sheds light on new molecular mechanisms by providing a list of variants and candidate genes associated with teratozoospermia in the Greek population.

Male infertility and the impact of lifestyle in the Greek population: A case-control study.

Background and Aims: Collection of epidemiological data has become a crucial step in every fertility evaluation, especially regarding idiopathic male infertility. Information on data such as tobacco smoking, alcohol intake, and body mass index can provide crucial information regarding the dynamics between fertility status and everyday practices. We aim to set the base for epidemiological studies on male infertility in the Greek population. Methods: Four hundred and fourteen Greek volunteers were asked to fill in a questionnaire regarding their characteristics and lifestyle preferences, followed by a seminogram. Depending on their answers, they were divided into groups and data were analyzed for correlation with seminogram parameters using Spearman's rank correlation test. Results: Our results indicate that a high body mass index (BMI) is negatively correlated with all three seminogram parameters (number, motility, and morphology) and exposure to radiation or chemicals is negatively correlated with sperm motility, with a p < 0.01. Conclusions: These findings indicate negative correlations of BMI and exposure to radiation/chemicals with semen parameters in the Greek population. Such information can be used to plan a diagnostic approach or even therapeutic interventions.

Accumulation of oocytes and/or embryos by vitrification: a new strategy for managing poor responder patients undergoing pre implantation diagnosis.

BACKGROUND: Low (or poor) responder patients are women who require large doses of stimulation medications and produce less than an optimal number of oocytes during IVF cycles. Low responder patients produce few oocytes and embryos, which significantly reduces their chances for success in a preimplantation genetic diagnosis (PGD) cycle. Accumulation of vitrified oocytes or embryos before the actual PGD cycle is a possible strategy that might increase patient's chances for a healthy pregnancy. AIM OF THE STUDY: This retrospective study evaluates the efficacy of a PGD program in low responder patients after repeated ovarian stimulation cycles with cumulative vitrification of oocytes and embryos. METHODS: Over a period of 30 months, 13 patients entering the PGD program were identified as poor responders after their first ovarian stimulation. These patients started a PGD cycle for one of the following indications: history of recurrent implantation failure (n=1), cystic fibrosis (n=1), X-linked microtubular myopathy (n=1), recurrent miscarriages (n=5), Duchene muscular dystrophy (n=1), chromosomal translocation (n=1) and high sperm aneuploidy (n=1).  After multiple ovarian hormonal stimulations patients had either all mature oocytes (Group A; 3 patients) or all of their day 2 embryos vitrified (group B; 10 patients). Mean total number of oocyte collections per patient was 2.3 (range: 2 - 5 cycles). RESULTS: In the actual PGD cycle, all vitrified oocytes from group A patients were warmed and underwent intra cytoplasmic sperm injection (ICSI) followed by culture up to day 3. For group B patients all vitrified day 2 embryos were warmed and cultured overnight. On day 3 of culture, all embryos from Group A and B had blastomere biopsy followed by genetic analysis. In group A, 20 embryos were found suitable for biopsy and genetic analysis; at least one healthy embryo was available for transfer for each patient.  For group B, 72 embryos in total were available for biopsy and PGD.  All patients, except one, had at least one healthy day 5 embryo for transfer (mean number of 2.1 embryos per transfer). Nine patients had a clinical pregnancy; 7 patients delivered a healthy baby. CONCLUSION: Low responder patients entering a PGD program might increase their chances for a healthy pregnancy by repeat ovarian stimulation in combination with cumulative oocyte or embryo vitrification.