Sperm Fertility Research Articles

SP22 sperm protein as a potential biomarker of fertility in humans: A preliminary study.

Infertility affects approximately 15% of couples of reproductive age, and 50% of the cases are directly related to men. The evaluation of male fertility is based on analyses of routine seminal parameters and the use of more advanced techniques can help identify fertility biomarkers. SP22 sperm protein is considered a biomarker in murine species since its concentration is highly correlated with sperm fertility. As the role of this protein as a biomarker is already well-established in other species, we hypothesized that this same correlation could apply to human. Thus, the present study aimed to investigate possible correlations between SP22 concentration and sperm parameters in fertile and infertile men. For this, a study was carried out on 21 volunteers' seminal samples who were grouped according to fertility as fertile (n=10) or infertile (n=11). Conventional and functional sperm analyses, membrane protein extraction, quantification and immunolocalization of SP22 were performed. The infertile volunteers showed an increase in the percentage of sperm with abnormalities in head morphology and a decrease in the percentage of sperm with intact plasma membrane and damaged acrosomal membrane. Serum concentration of the hormone SHBG was also decreased in infertile volunteers. The damage to the plasma membrane was positively correlated with the superoxide anion production. Although none of the functional parameters were correlated with SP22 concentration, type D sperm motility was negatively correlated and type A+B sperm motility was positively correlated. This preliminary study opens new paths in the characterization of SP22 as a non-invasive biomarker for predicting fertility/infertility.

MP30-07 EVALUATION OF MTESE OUTCOMES IN PATIENTS WITH PARTIAL AZFb Y CHROMOSOME MICRODELETIONS

You have accessJournal of UrologyCME1 Apr 2023MP30-07 EVALUATION OF MTESE OUTCOMES IN PATIENTS WITH PARTIAL AZFb Y CHROMOSOME MICRODELETIONS Jonathan Gal, Jessica Marinaro, Anna Mielnik, and Peter Schlegel Jonathan GalJonathan Gal More articles by this author , Jessica MarinaroJessica Marinaro More articles by this author , Anna MielnikAnna Mielnik More articles by this author , and Peter SchlegelPeter Schlegel More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Y chromosome microdeletions (YCMD) are a common cause of non-obstructive azoospermia. While men with deletions in the azoospermia factor C (AZFc) region have a high likelihood of having sperm in the ejaculate, or sperm retrievable with effective microdissection testicular sperm extraction (mTESE) procedure, those with complete deletions in the AZFa or AZFb regions have a poor prognosis for identification of sperm. Men with AZFa deletions typically do not have any germ cells, and no men, to-date, with complete AZFb deletions have had usable sperm identified. Partial deletions of AZFb appear to portend a different prognosis for fertility potential. Our objective was to evaluate semen parameters, sperm retrieval rates and fertility outcomes in men with partial deletions of the AZFb region. METHODS: We retrospectively reviewed 468 men with YCMDs who underwent testing at our institution from 4/1996 to 8/2022. Of these, we identified 20 men with partial AZFb deletions, defined as a deletion of some but not all tested segments from sY1227 to sY1291. Of these, 8 had documented semen analysis and underwent mTESE by a single, high-volume surgeon (PS). Sperm retrieval rate (SRR) was defined as the presence of mature sperm identified after processing by a trained andrologist. RESULTS: Of the 8 men with a partial AZFb deletion, 7 (87.5%) had a sY117-sY1291 deletion, and one (12.5%) had an sY1227-sY1192 deletion. All 8 men (100%) were azoospermic. Mean testosterone levels were 450.5 ± 350.5 (ng/dL), Mean FSH 6.5±2.8 (mIU/mL). After mTESE, 1 of 7 men (14%) with an sY117-sY1291 deletion was found to have sperm, and the 1 man with a sY1227-sY1192 deletion was found to have round spermatids on histology. Overall, the SRR was 12%. All pathology specimens (n=8, 100%) demonstrated maturation arrest. CONCLUSIONS: In this large series, we demonstrate that while patients with partial AZFb deletions are azoospermic, it is possible to find sperm at least for some men with mTESE. Distinguishing partial from complete AZFb deletions may have prognostic value. Further work is needed to determine which men with partial AZFb deletions are more likely to have a successful mTESE procedure. Source of Funding: Author JG is supported in part by the Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e393 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jonathan Gal More articles by this author Jessica Marinaro More articles by this author Anna Mielnik More articles by this author Peter Schlegel More articles by this author Expand All Advertisement PDF downloadLoading ...

Complications in pregnancies achieved by assisted reproduction.

Pregnancies resulting from assisted reproductive technologies (ART) have been documented to have a higher risk of adverse effects. To provide evidence on obstetric and perinatal complications associated with conceptions by ART versus spontaneous pregnancies. Comprehensive review of original articles published between 2010 and 2018 addressing the more common obstetric and perinatal complications in pregnancies resulting from in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), in comparison with spontaneous conceptions. Thirty-seven original articles, which reported on 26 cohort studies and 11 case-control trials, were included. IVF and ICSI conceptions were associated with a larger number of obstetric and perinatal complications such as low birth weight, prematurity, low weight for gestational age, admission to the neonatal intensive care unit, congenital malformations, C-sectionand premature rupture of membranes, among others. Pregnancies by ART are associated with an increased risk of obstetric and perinatal complications in comparison with spontaneous conceptions. Further research is needed to determine which aspects result in higher risk.

CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa.

The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion.

Cryopreservation of sperm in the spotted scat, Scatophagus argus (Linnaeus, 1766) - Conservation and aquaculture perspectives

This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.

Human Leukocyte Antigen Alleles Compatibility and Immunophenotypic Profile Associations in Infertile Couples.

The maternal immune system has a major role in the successful embryo implantation and maintenance of the pregnancy. This study aimed to investigate the maternal immunophenotyping profile (percentage of Natural Killer [NK] cellsand the CD4/CD8 [cluster designation] ratio in peripheral blood lymphocytes)and the HLA (Human Leukocyte Antigen)-DQA1 alleles sharing in infertile couples. This cross-sectional study included 78 women who had experienced at least two spontaneous miscarriages and 110 women with a history of recurrent implantation failures after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) (IVF-ET failures). The NK cell percentage and the CD4/CD8 ratio were determined by flow cytometry. Genotyping of the HLA-DQA1alleles was carried out for all women and their partners, and couple HLA-DQA1 compatibility was expressed as the percentage of commonHLA-DQA1 alleles (totaling 35 alleles)shared between spouses to the sum of the unique alleles observed. In women with recurrent miscarriages, high values (%) of the NK population with a median (interquartile range [IQR]) of 10.3% (7.7% to 12.5%) and CD4/CD8 ratio (1.7) (1.5 to 2.1) were found. In women with IVF-ET failures, the (%) NK population (10.5%) (8.6% to 12.5%) and CD4/CD8 ratio (1.8) (1.5 to 2.1) were similarly increased (p=0.390, and p=0.490, respectively). The proportion of women with >10% NK cells was 53.8% and 58.2% in women with miscarriages and IVF-ET failures, respectively (p=0.554). The prevalence of HLA-DQA1*5 allele carriage was elevated in women with miscarriages as well as those with IVF-ET failures (52.6% and 61.8%, respectively; p=0.206). The proportion of couples with high (>50%) HLA-DQA1 sharing was 65.4% in the group with miscarriages and 73.6% in the group with IVF-ET failures, respectively (p=0.222). The CD4/CD8 ratio was statistically significantly positively correlated with the (%) NK population in women with IVF-ET failures (rho = 0.297, p=0.002) and with the (%) HLA-DQA1 sharing in the group with miscarriages (rho = 0.266, p=0.019). The couples in which both spouses were carriers of the HLA-DQA1*5 allele had an increased probability of high (>50%) HLA-DQA1 compatibilitycompared with the couples in which neither of the spouses carried the allelein the miscarriage group (OR = 24.3, 95% CI: 3.0 to 198.9, p<0.001), and the IVF-ET failure group (OR = 10.5, 95% CI: 2.2 to 49.8, p<0.001). The peripheral NK (%) population and CD4/CD8 ratio, as well as the prevalence of the HLA-DQA1*5 allele, were elevated in women with recurrent miscarriages and IVF-ET failures. Furthermore, these couples with negative reproductive outcomes had a high percentage of HLA-DQA1 allele similarity. The presence of the HLA-DQA1*5 allele in spouses was strongly associated with overall couple HLA-DQA1 compatibility, implying that it could be used as a surrogate marker for assessing overall immunological compatibility in infertile couples.

Effects of preconception exposure to phthalates on mouse sperm capacitation parameters.

Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. Adult male C57BL/6Jmice aged 8-9weeks were exposed to di(2-ethylhexyl) phthalate, di-n-butyl phthalate, or their mixture (di-n-butyl phthalate+di(2-ethylhexyl) phthalate) at 2.5mg/kg/day or vehicle for 40days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer-assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2-ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa.

Semen quality analysis of the Airshire bulls in relation to the AH1 fertility haplotype

The results of the studies (2019-2021) of increased incidence of genetic mutations, such as fertility haplotypes, in the population that result in lower reproductive rates in dairy cattle are presented. Semen quality of the Ayrshire breed bulls was evaluated for motility and morphological indices due to rs475678587 in the UBE3B gene locus associated with the AH1 fertility haplotype. Semen samples (n = 14) were obtained from the Center of Collective Use "Genetic Semen Collection of Domestic and Foreign Cattle Breeds" of the All-Russian Research Institute of Farm Animal Genetics and Breeding (ARRIFAGB) and belonged to the animals born in 2014 and older (average age of bulls was 6-15 years). Genotyping for rs475678587 of the UBE3B gene was performed by Sanger sequencing. Animals with the heterozygous CT genotype were defined as carriers of the AH1 haplotype (AH1-C), and those with the homozygous CC genotype were defined as free of the AH1 haplotype (AH1-F). Frozen-thawed semen was evaluated according to the following parameters: total and progressive motility, percentage of normal spermatozoa. Morphological indices of sperm quality were assessed after differential staining of cellular elements of spermatozoa using a commercial kit "Diakhim-Diff Quick" (ABRIS+ scientific and production company) in sperm smears. The following indicators were taken into account: acrosomal abnormalities, abnormalities in the head, tail, and neck of the spermatozoon. Data on the indicators of native semen (motility, concentration), obtained immediately after collection, were received from the archive of the technical service of the breeding enterprise. The results of the studies have shown that according to physiological and morphological indices the semen obtained from the bulls carrying the haplotype AN1 did not differ significantly from those of the bulls free of the mutation. In terms of total motility, the semen of the haplotype fertility carrier bulls was almost 2% higher than that of the haplotype free bulls. It can be noted that the haplotype carriers of fertility bulls were generally characterized by good sperm fertility indices.

Spontaneous pregnancies post-microsurgical varicocelectomy in infertile men with severe oligozoospermia: a preliminary vietnamese report.

This study evaluated whether microsurgical varico-celectomy performed in infertile men with severe oligozoospermia (SO) resulted in improved semen parameters or increased rates of spontaneous pregnancy (SP) and performed a cost-effectiveness analysis comparing intrauterine insemination (IUI), in vitro fertilization (IVF), and varicocelectomy. This study included 25 patients with SO who underwent microsurgical varicocelectomy between September 2019 and May 2022, which resulted in post-surgical SP in all cases. Men with azoospermia, abnormal karyotype, or Y-chromosome microdeletion were excluded from the study. Serum luteinizing, follicle-stimulating, and testosterone hormones were measured preoperatively. Semen was analyzed every 3 months postoperation. The incidence of SP was recorded at each visit. Cost-effectiveness for assisted reproductive technologies was calculated based on reported costs. Several parameters were evaluated as potential predictors of the response to microsurgical varicocelectomy using univariate and multivariate analyses. After a mean postoperative observation period of 7 months, 25 couples with SP after microsurgical varicocelectomy were recruited. The mean sperm concentration increased from 3 million/mL (interquartile range [IQR]: 2-5 million/mL) to 12 million/mL (IQR: 5-17 million/mL; p<0.05), and mean sperm motility improved from 4% (IQR: 3%-6%) to 7.6% (p<0.05). Total motile sperm count (TMSC) increased to 3.08 million (IQR: 1.02-5.83 million) from a preoperative value of 0.34 million (IQR: 0.16-0.83 million). A cost-effectiveness analysis comparing IVF with varicocelectomy indicates that varicocelectomy may represent a better first-line option for infertile men with very low preoperative TMSC. However, further research remains necessary to confirm this result. Varicocelectomy should be discussed as a treatment option for men with SO and may improve sperm quality and fertility potential, resulting in SP.

Sperm Quality Assessment of White Shrimp (Litopenaeus Vannamei) Broodstock using Comet Assay

The quality of sperms is considered one of the factors determining fertilization success of white shrimp, Litopenaeus vannamei. Generally, conventional methods such as sperm count and viability are used for determining sperm quality. Occasionally, the fertilization rate does not correlate with what conventional parameters indicate. Presently, it has become increasingly evident that sperm DNA damage is a potential cause of an unexplained fertilization incompetence of the sperm. Therefore, the present study aimed to determine the feasible application of the comet assay for measuring sperm quality from different male broodstock and to analyze the correlation between sperm DNA integrity and fertilization efficacy of the shrimp. Male and female shrimp sampled from 4 different broodstock ponds were mated in experimental tanks (n = 5). Sperm were collected and subjected to alkaline comet assay. Sperm count, sperm viability, and Gonadosomatic index(GSI) were analyzed for comparison. Viability of the obtained nauplii were recorded and used as larvae production efficiency. The results revealed that nauplius numbers were significantly different among treatments (6.78±1.61×104, 12.18±1.17×104, 16.76±0.97×104 and 21.38±1.14×104 cells/spawn in treatment 1, 2, 3 and 4, respectively), indicating the different fertilization rate between treatments. The average body weights, lengths, and GSIs of broodstock were not significantly different, indicating that the morphological factors were not major causes of the different nauplii production among treatments. The results of comet measurements clearly showed that the levels of comet parameters correlated with the numbers of nauplii obtained from each treatment while the conventional sperm analyses were unable to detect the different quality of the sperm among treatments. This indicates that the degree of sperm DNA damage correlates with the fertilization rate of the shrimp and can be used as a sensitive indicator for determining sperm quality of broodstock shrimp. HIGHLIGHTS Sperm DNA damage is one of the potential causes of low fertilization rate in L. vannamei captive breeding Comet assay is used to assess the degree of DNA damage in a single cell such as shrimp sperm cell using electrophoresis technique Comet assay is more sensitive to detect the sperm integrity among individual L. vannamei broodstock when compared to morphological methods Sperm DNA damage can be used as a sensitive indicator for determining the sperm quality of L. Vannamei GRAPHICAL ABSTRACT

A Longitudinal Characterization of the Seminal Microbiota and Antibiotic Resistance in Yearling Beef Bulls Subjected to Different Rates of Gain.

In this study, we evaluated the seminal and fecal microbiota in yearling beef bulls fed a common diet to achieve moderate (1.13 kg/day) or high (1.80 kg/day) rates of weight gain. Semen samples were collected on days 0 and 112 of dietary intervention (n = 19/group) as well as postbreeding (n = 6/group) using electroejaculation, and the microbiota was assessed using 16S rRNA gene sequencing, quantitative PCR (qPCR), and culturing. The fecal microbiota was also evaluated, and its similarity with seminal microbiota was assessed. A subset of seminal bacterial isolates (n = 33) was screened for resistance against 28 antibiotics. A complex and dynamic microbiota was detected in bovine semen, and the community structure was affected by sampling time (R2 = 0.16, P < 0.001). Microbial richness increased significantly from day 0 to day 112, and diversity increased after breeding (P > 0.05). Seminal microbiota remained unaffected by the differential rates of gain, and its overall composition was distinct from fecal microbiota, with only 6% of the taxa shared between them. A total of 364 isolates from 49 different genera were recovered under aerobic and anaerobic culturing. Among these seminal isolates were pathogenic species and those resistant to several antibiotics. Overall, our results suggest that bovine semen harbors a rich and complex microbiota which changes over time and during the breeding season but appears to be resilient to differential gains achieved via a common diet. Seminal microbiota is distinct from the fecal microbiota and harbors potentially pathogenic and antibiotic-resistant bacterial species. IMPORTANCE Increasing evidence from human and other animal species supports the existence of a commensal microbiota in semen and that this seminal microbiota may influence not only sperm quality and fertility but also female reproduction. Seminal microbiota in bulls and its evolution and factors shaping this community, however, remain largely underexplored. In this study, we characterized the seminal microbiota of yearling beef bulls and its response to the bull age, different weight gains, and mating activity. We compared bacterial composition between seminal and fecal microbiota and evaluated the diversity of culturable seminal bacteria and their antimicrobial resistance. Our results obtained from sequencing, culturing, and antibiotic susceptibility testing provide novel information on the taxonomic composition, evolution, and factors shaping the seminal microbiota of yearling beef bulls. This information will serve as an important basis for further understanding of the seminal microbiome and its involvement in reproductive health and fertility in cattle.

Solid storage supplemented with serine of rooster semen enhances higher sperm quality and fertility potential during storage at 5°C for up to 120 h

Information on prolonging the storage duration of cold semen with acceptable fertility in roosters is limited. This study aimed to determine the efficiency of solid storage with the addition of various concentrations of serine to the Thai native rooster (Pradu Hang Dum) semen extender on semen quality and fertility potential during storage at 5°C for up to 120 h. Pooled semen was diluted with a base extender and a gelatin extender containing 0, 2, 4, and 6 mM serine, then stored at 5°C for 120 h. In Experiment 1, the semen quality and malondialdehyde (MDA) concentrations were assessed at 0, 24, 72, and 120 h after storage. In Experiment 2, fertility potential in terms of fertility and hatchability rates was determined using the most effective solid-storage semen from Experiment 1. Sperm quality decreased with increasing storage time (P < 0.05). The lowest semen quality was observed in the control group since T24 of storage compared with the other groups (P < 0.05). Progressive motility, viability, and mitochondrial function were higher (P < 0.05) in the extender supplemented with gelatin and serine groups than those in the gelatin alone group at T72 and T120. In the extender supplemented with gelatin and serine groups, the highest semen quality was observed in the gelatin with 4 mM serine groups. The differences among extenders supplemented with serine were insignificant (P > 0.05), and the lowest MDA was observed in the gelatin with 4 mM serine groups. The fertility and hatchability rates in gelatin with 4 mM serine at T24 were comparable to those in fresh semen (83.87 and 86.12% vs. 86.66 and 88.3%; P > 0.05). Those of T72 were significantly better than those of the control at the same hour of storage (64.08 and 71.61% vs. 52.38 and 64.48%), while those of T120 were not different among groups. In summary, a semen extender as a solid medium supplemented with 4 mM serine successfully preserved the rooster semen for a long duration up to 72 h of storage time.

A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori.

Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.

Time-resolved ICP-MS analysis of mineral element contents and distribution patterns in spermatogenic cells of different types

Mineral elements play an important role in the spermatogenesis, maturation, and fertilization of sperm. It is of great scientific significance to study the role of mineral elements in spermatogenesis by accurately measuring the content of elements in different spermatogenic cells and analyzing the distribution pattern of elements in spermatogenesis. Here, time-resolved inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the content and distribution patterns of mineral elements in spermatogenic cells of different types at the single cell level. Firstly, spermatogonia, spermatocytes, round spermatids and elongating spermatids were successfully isolated from testis of mice of different weeks of age by differential adherent method and discontinuous bovine serum albumin (BSA) density gradient method. Then, signal profiles and elemental distributions of 24Mg, 31P, 52Cr, 55Mn, 56Fe and 66Zn in spermatogenic cells were obtained with dwell time at 0.1 ms. Based on the results of acid digestion, we derived a formula to calculate element content in single cell from peak area for each element, and the feasibility and universality of the formula in the quantitative detection of single cell elements were verified by sperm samples to a certain extent. The detection results of element content in single cell showed that the content of 31P in elongating spermatids was significantly higher than that in spermatogonia, spermatocytes and round spermatids (P < 0.01), and the distribution range was wider. However, the 52Cr and 56Fe content of elongating spermatids was lower than that of spermatogonia, spermatocytes and round spermatids (P < 0.05). When spermatogonia developed into round spermatids, the contents of 55Mn and 66Zn in single cell increased significantly (P < 0.05), then decreased to the lowest in elongating spermatids. In addition, the significant decrease of 52Cr, 55Mn, 56Fe and 66Zn content in elongating spermatids also be visually observed from the center of the fitting curve of the element signal intensity distribution moving to the left. This study provides an elemental view of the changes in elemental content at various stages of spermatogenesis at the single-cell level. Time-resolved ICP-MS is used to detect mineral elements content and distribution patterns in spermatogenic cells of testis, which is helpful to better explore the stages and modes of action of various elements in spermatogenesis, and provide direct evidence for revealing the effects of element content changes on spermatogenesis and semen quality regulation.

Chronic poor healing wounds of post cesarean scar diverticulum: Altered angiogenesis and immunobiology

Cesarean section (CS) scar diverticula (CSD) is an important cause impede further fecundity, with rather complicated pathophysiologic mechanisms and unclear etiopathogenesis. In this study, we detect the influences of CSD on the pregnancy outcomes in in-vitro fertilization (IVF) or intra-cytoplasmic sperm injection (ICSI) embryo transfer (ET) cycles, and further explore the mechanisms involved based on histopathology and immunology differences in endometrium between CSD and vaginal birth (VB) women. The CS group had significantly lower CLBR compared to NCS group. Histopathological analysis showed that the higher prevalent of CE is accompanied by excessive fibroblast proliferation at the lower segment of uterus and significantly exaggerated vascular proliferation in situ. Intrauterine inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, TNF-α and SDF-1α were also increased in CSD group. The present data suggests impaired fecundity in CSD women undergoing IVF/ICSI treatment. Although the causal relationship is ambiguous, the potential mechanisms may involve persistent inflammatory response in the uterine cavity, active vascular proliferation accompanied with increased fibrosis which are responsible for poor chronic wound healing of CSD.

Concepts to Consider When Choosing an Assay to Predict Potentially Fertile Sperm

The primary goal of semen analysis is to determine whether or not an ejaculate has a sufficient number of potentially fertile sperm to achieve the desired outcome of conception. Given the significant advancements in Assisted Reproductive Technology (ART) in developed countries where IVF and ICSI are widely available to address male-factor fertility concerns, then what is the remaining utility of semen analysis? For many physicians and patients, assays to evaluate the fertilization potential of semen continue to be essential as a gateway to specialists who can perform the aforementioned ART procedures. Most initial semen analysis requests are from gynecologists, general practitioners, family practitioners, and urologists who wish to advise their patients regarding their fertility status. If male-factor infertility is detected, only then can these physicians refer patients to reproductive endocrinologists, often due to insurance considerations. Infertility is also a global concern, and many doctors and patients worldwide do not have access to the advanced technologies that are available in wealthy, developed nations. For these patients, knowing the fertility status of the man is necessary in order to overcome infertility.

Transcriptome identification of genes associated with uterus–vagina junction epithelial folds formation in chicken hens

The development regulation of the uterine-vaginal junction (UVJ) epithelial folds during the sexual maturation of female birds played crucial roles in the adults' sperm storage duration and fertilization capability. However, there is a lack of studies on it in the breeding field of laying hens. In this study, White Leghorn was used for the morphological and developmental studies. According to the morphological characteristics, the development of the UVJ epithelial folds was classified into 4 stages (morphological stage T1-T4). Significant individual differences were observed simultaneously, which is one of the factors leading to the adults' UVJ morphological differences. Bulk RNA-seq suggested the different regulations of UVJ epithelial folds were classified into 3 stages (developmental stage S1-S3). Genes enriched in cell proliferation, differentiation, polarity, migration, adhesion and junction were supposed to regulate UVJ epithelial fold formation. Single-cell RNA-sequencing (scRNA-seq) showed significant differences between different types of cells within UVJ at the developmental stage S2. Immunohistochemical studies confirmed that the different proliferation rates between the epithelium and nonepithelium were one of the key factors leading to the formation of UVJ epithelial folds. Genes in the TGF-beta and WNT pathways may play roles in regulating the proliferation and differentiation of epithelium. Some factors, such as CHD2, CDC42, and carbonic anhydrases, were important participants in forming UVJ epithelial folds. This study will provide new thoughts on the differential regulation of fertilization traits from the developmental biology perspective.

Inclusion of Spirulina platensis and Salvia verbenaca extracts to boost semen quality and fertilization ability in sheep.

The aim of this study was to investigate the effect of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to skimmed milk (SM) extender on ram sperm quality and fertility. Semen was collected using an artificial vagina, extended in SM to reach a final concentration of 0.8 × 109 spermatozoa/mL, stored at 4°C and evaluated at 0, 5H and 24 H. The experiment has been performed in three steps. Firstly, from four extracts (methanol: MeOH, acetone: Ac, ethyl acetate: EtOAc and hexane: Hex) of SP and SV, only acetonic and hexanoic extracts of SP and acetonic and methanolic extracts of SV showed the highest in vitro antioxidant activities and were then selected for the following step. Thereafter, the effects of four concentrations (1.25, 3.75, 6.25, and 8.75 μg/mL) of each selected extract on stored sperm motility were evaluated. The output of this trial led to select the best concentrations having beneficial effects on sperm quality parameters (viability, abnormalities, membrane integrity, and lipid peroxidation) and fertility after insemination. The results showed that the same concentration (1.25 μg/mL) of both Ac-SP and Hex-SP, as well as 3.75 μg/mL of Ac-SV and 6.25 μg/mL of MeOH-SV, maintain all sperm quality parameters at 4°C during 24 H of storage. Besides, no difference was found in fertility between the selected extracts and the control. In conclusion, SP and SV extracts were shown to improve the quality of ram sperm and to maintain fertility rate after insemination as similar or competitive to many previous studies published in the field.

Seminal Extracellular Vesicles and Their Involvement in Male (In)Fertility: A Systematic Review.

Seminal plasma contains numerous extracellular vesicles (sEVs). Since sEVs are apparently involved in male (in)fertility, this systematic review focused on studies specifically investigating such relationship. Embase, PubMed, and Scopus databases were searched up to 31 December 2022, primarily identifying a total of 1440 articles. After processing for screening and eligibility, 305 studies were selected as they focused on sEVs, and 42 of them were considered eligible because they included the word fertility or a related word such as infertility, subfertility, fertilization, and recurrent pregnancy loss in the title, objective(s), and/or keywords. Only nine of them met the inclusion criteria, namely (a) conducting experiments aimed at associating sEVs with fertility concerns and (b) isolating and adequately characterizing sEVs. Six studies were conducted on humans, two on laboratory animals, and one on livestock. The studies highlighted some sEV molecules, specifically proteins and small non-coding RNAs, that showed differences between fertile and subfertile or infertile males. The content of sEVs was also related to sperm fertilizing capacity, embryo development, and implantation. Bioinformatic analysis revealed that several of the highlighted sEV fertility-related proteins would be cross-linked to each other and involved in biological pathways related to (i) EV release and loading and (ii) plasma membrane organization.

Integration of omics studies indicates that species-dependent molecular mechanisms govern male fertility

BackgroundComparative and comprehensive omics studies have recently been conducted to provide a comprehensive understanding of the biological mechanisms underlying infertility. However, because these huge omics datasets often contain irrelevant information, editing strategies for summarizing and filtering the data are necessary prerequisite steps for identifying biomarkers of male fertility. Here, we attempted to integrate omics data from spermatozoa with normal and below-normal fertility from boars and bulls, including transcriptomic, proteomic, and metabolomic data. Pathway enrichment analysis was conducted and visualized using g:Profiler, Cytoscape, EnrichmentMap, and AutoAnnotation to determine fertility-related biological functions according to species.ResultsIn particular, gamete production and protein biogenesis-associated pathways were enriched in bull spermatozoa with below-normal fertility, whereas mitochondrial-associated metabolic pathways were enriched in boar spermatozoa with normal fertility. These results indicate that below-normal fertility may be determined by aberrant regulation of protein synthesis during spermatogenesis, and the modulation of reactive oxygen species generation to maintain capacitation and the acrosome reaction governs boar sperm fertility.ConclusionOverall, this approach demonstrated that distinct molecular pathways drive sperm fertility in mammals in a species-dependent manner. Moreover, we anticipate that searching for species-specific signaling pathways may aid in the discovery of fertility-related biomarkers within large omics datasets.