Single Sperm Research Articles

Long-read-based single sperm genome sequencing for chromosome-wide haplotype phasing of both SNPs and SVs

Long-read-based single sperm genome sequencing for chromosome-wide haplotype phasing of both SNPs and SVs

Exogenous Metals Atlas in Spermatozoa at Single-Cell Resolution in Relation to Human Semen Quality

While exogenous metal/metalloid (metal) exposure has been associated with reduced human semen quality, no study has assessed the associations of exogenous metals in human spermatozoa with semen quality. Here, we developed a strategy to explore the associations between exogenous metals in spermatozoa at single-cell resolution and human semen quality among 84 men screened as sperm donors, who provided 266 semen samples within 90 days. A cellular atlas of exogenous metals at the single-cell level was created with mass cytometry (CyTOF) technology, which concurrently displayed 18 metals in more than 50 000 single sperm. Exogenous metals in spermatozoa at single-cell resolution were extremely heterogeneous and diverse. Further analysis using multivariable linear regression and linear mixed-effects models revealed that the heterogeneity and prevalence of the exogenous metals at single-cell resolution were associated with semen quality. The heterogeneity of lead (Pb), tin (Sn), yttrium (Y), and zirconium (Zr) was negatively associated with sperm concentration and count, while their prevalence showed positive associations. These findings revealed that the heterogeneous properties of exogenous metals in spermatozoa were associated with human semen quality, highlighting the importance of assessing exogenous metals in spermatozoa at single-cell resolution to evaluate male reproductive health risk precisely.

Higher incidence of preeclampsia among participants undergoing in-vitro fertilization after fewer sperm exposures

ObjectiveEvaluation of preeclampsia (PE) incidence among participants undergoing in vitro fertilization (IVF) after various cycles of sperm donation (SD) via intrauterine inseminations (IUI) or IVF. Study designA retrospective case-control study was conducted at a single tertiary medical center between 2011 and 2019 which included participants who conceived via IVF using SD from a single sperm bank and had a successful singleton birth at Sheba Medical Center. The study cohort was divided into two groups: Group 1 (participants who conceived via IVF after 0–1 cycles of IUI or IVF from the same sperm donor) and Group 2 (participants who conceived via IVF after 2 or more cycles of IUI or IVF from the same sperm donor).Baseline characteristics and pregnancy outcomes between the two groups were compared. In addition, a comparison between the study groups and a control of participants of the same age who conceived spontaneously and had a singleton birth at Sheba Medical Center during the same period with a record of up to two previous deliveries was done. ResultsA total of 228 participants conceived through IVF from SD and met the inclusion criteria. Of these, 110 were defined as Group 1 and 118 as Group 2. The participants showed no differences in their age, gravidity and parity, chronic medical conditions, or history of pregnancy complications.Preeclampsia was positively associated with Group 1 (9 [8.2%] vs. 2 [1.7%], P = 0.022).PE was observed to be more prevalent in Group 1 (P < 0.001) when compared to a control group of 45,278 participants who conceived spontaneously. No significant differences were observed in comparing Group 2 with the same control group. ConclusionThe incidence of PE was higher among participants who were exposed to 0–1 IUI or IVF cycles than in those who were exposed to 2 or more cycles of IUI or IVF from the same sperm donor. On comparing both groups with a control group, the incidence of PE was higher in participants who were exposed to 0–1 cycles, while there was no difference in participants exposed to 2 or more cycles. Implications statementIf there is a statistically significant increase in the incidence of PE when conception occurred following fewer sperm exposures, then there may be a correlation between these two. The reason for this is not entirely clear, but based on former literature, we hypothesize it may be related to the fact that repeated exposures to paternal antigens may alter the maternal immune response causing a better adaptation to the semi-allogenic nature of the fetus, its paternal half.

A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system)

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

Gender Preselection in the Buffalo Species (Bubalus Bubalis): Preliminary Data on Different Strategies and Pregnancy Outcome

In this study, a new comparison was tested in Mediterranean Italian buffaloes (Bubalus bubalis), between two different strategies for obtaining sperm cells of the desired sex. In addition, a control group of animals was included in the study, where only ordinary semen straws, containing unselected sper cells were used. Sexed and control straws were then used for Artificial Insemination (AI) in order to confront pregnancy and gender rates. The first of the two strategies to preselect sperm cells relies on the physical determination of DNA content in single spermatozoa though cell sorting machines. The second strategy instead, relies on the coincubation of either fresh or frozen/thawed semen with a specific solution specifically designed for the species of interest. The trial involved a total of 206 heifers and 211 pluriparous buffaloes belonging to a buffalo farm in the south of the Lazio Region in Italy. In this study, no significant differences between treated and control groups were detected in terms of pregnancy rates both on heifers (P=0.6) and pluriparous buffaloes (P=7). With regard to gender rates, pregnancy rates were significantly different among the three groups of heifers (P=0.001) and the three groups of pluriparous buffaloes (P=0.002). Sperm cells preselected for gender determination through Cell Sorting (CST) resulted effective in shifting the gender ratio towards the female, when compared to both the Coinbubation Technique (COiT) and control group of animals.

Application of single sperm sequencing for the preimplantation genetic testing of a Chinese family affected with Spinal muscular atrophy

To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.

TMT-based quantitative proteomics analysis reveals the differential proteins between fresh and frozen-thawed sperm of yak (Bos grunniens)

Sperm cryopreservation is one of the most effective methods for the conservation of germplasm resources and used of superior sires widely. However, the motility of yak (Bos grunniens) sperm was low after thawing and the proteomics changes in sperm cryopreservation remain unknown. Therefore, the aim of this study was to explore the differences between fresh sperm and frozen sperm of yak through the proteomic analysis and thus improve the understanding of sperm cryodamage. The Tandem Mass Tags (TMT) technology was used to screen differentially expressed proteins (DEPs) before and after freezing. Then, GO and KEGG analysis were conducted to analyze the DEPs enriched signaling pathways. Finally, the DEPs, including superoxide dismutase 1 (SOD1) and NADH ubiquinone oxidoreductase core subunit S8 (NDUFS8) were verified by the immunofluorescence technique. The results showed that there were 229 DEPs between fresh and frozen-thawed yak sperm. Compared with the fresh sperm, 120 proteins were up-regulated and 109 proteins were down-regulated in frozen-thawed sperm. The GO annotation showed that the up-regulated proteins enriched in metabolic and cytoskeleton-related processes, including lipoprotein metabolic process, lipid transport, extracellular region and intermediate filament cytoskeleton organization. In contrast, the down-regulated proteins enriched in biological processes including single fertilization, sperm capacitation and response to unfolded protein. KEGG pathway analysis indicated that freezing and thawing affected the oxidative phosphorylation pathway, the fructose and mannose metabolic pathway and the glycerolipid metabolic pathway of yak sperm. Immunofluorescence results showed that the protein expression level of SOD1 protein in the frozen group was significantly lower than that in the fresh group (P < 0.01), and the protein expression level of NDUFS8 protein was significantly higher in frozen group (P < 0.01). This study revealed the DEPs between fresh and frozen-thawed sperm and provides a theoretical basis to further explore the exertion of normal biological functions of yak sperm after freezing and thawing.

True Nodes of the Umbilical Cords of a Mono-Amniotic Pregnancy

The incidence of twin pregnancies, has increased due to assisted reproductive technologies [1]. We note a doubling between 1970 and 2010 from less than 8‰ to nearly 16‰ of pregnancies (Pison et al., 2014; Pison et al., 2015) in developed countries [2] and 17‰ to 20‰ in sub-Saharan Africa. Monozygotic twins are the product of fertilization of a single egg by a single sperm, with the egg splitting into two within the first few days after fertilization. If the division takes place after the 8th day, the pregnancy is mono-chorionic mono-amniotic (less 1% of cases of monozygotic pregnancies) and represents 1 in 30,000 pregnancies. It is the development of two fetuses within the same amniotic sac, representing the rarest type of twin pregnancy (1% of monozygotic twin pregnancies) but being the one with the highest fetal and maternal morbidity. The main complication feared is coiling and knots of the umbilical cords responsible for abnormal heartbeat, fetal suffering and eventual fetal death. We report the case of in utero fetal death of twins from a mono choral mono amniotic pregnancy secondary to knots and coiling of the cord in order to discuss the diagnostic and therapeutic aspects.

Forensic Genetics and the Differentiation of Monozygotic Twins by Mitochondrial DNA Analysis

Forensic genetics uses STR markers as the gold standard for human identification. However, this technique does not allow the differentiation of monozygotic twins, since they originate from the fertilization of a single egg and a single sperm. Studies aiming to solve this problem have been developed and the complete sequencing of mitochondrial DNA has shown to be relevant. The present study aimed to verify the possibility of differentiating monozygotic twins through the analysis of the d-loop region of mitochondrial DNA, using the Sanger sequencing technique in blood and oral mucosa samples. Twelve pairs of volunteer monozygotic twins were selected, zygosity was confirmed by analyzing the genetic profile generated using STR markers. Regarding analyses of the d-loop region of mitochondrial DNA, a single pair of twins (gm46) showed a point heteroplasmy at position 16,290 (HV1). However, this finding was not able to differentiate between individuals of the same pair, since both showed the heteroplasmy in both biological samples (blood and oral mucosa). No other genetic variation was detected in the pairs of twins analyzed that could differentiate them. We suggest analyzing the mitochondrial DNA molecule as a whole in an attempt to identify a greater amount of potential variation by applying the Massive Parallel Sequencing technique.

Response to Zhang et al., 'do egg cell-secreted aspartic proteases promote gamete attachment?'

In angiosperms, the molecular mechanism controlling polytubey/polyspermy block and fertilization recovery was not unraveled until recently (Zhong et al., 2022), which can be described as follows: Five pollen tube-expressed RALF peptide ligands, perceived by the receptor complex composed of three receptors FER/ANJ/HERK1 at the septum, establish the polytubey block at the septum so that only one pollen tube is allowed to emerge from the septum to target the ovule for fertilization. Pollen tube rupture releases the two sperm cells and fertilization is completed within 20 minutes. This article is protected by copyright. All rights reserved.

Do egg cell‐secreted aspartic proteases promote gamete attachment?

During the double fertilization process of flowering plants (angiosperms), pollen tubes transport a pair of immobile sperm cells towards and inside the embryo sac for fusion with egg and central cell, respectively. Male-female gamete interactions during fertilization are critical for reproductive success and seed development, and thus have interested biologists since decades. Compared to animals, little is known about gamete interaction mechanisms in angiosperms. This article is protected by copyright. All rights reserved.

Laboratory and Clinical Outcomes of Single Sperm Cryopreservation in Patients Underwent Micro-TESE

Laboratory and Clinical Outcomes of Single Sperm Cryopreservation in Patients Underwent Micro-TESE

Bundles of Sperm: Structural Diversity in Scorpion Sperm Packages Illuminates Evolution of Insemination in an Ancient Lineage

The spermatozoa of scorpions are often bundled together, forming a type of sperm conjugation known as a sperm package. Sperm packages may be found inside the testes and seminal vesicles but vanish in the female atrium, leaving free spermatozoa. Previous studies, based on a limited number of taxa, suggested a diversity of sperm package morphology across the order Scorpiones C.L. Koch, 1850. However, the sperm packages of most scorpion taxa remained unknown. The present study provides the first systematic survey of sperm package morphology across the order, covering 89 exemplar species in 66 genera and 19 families representing all suprafamilial ranks, with a more detailed investigation of the family Bothriuridae Simon, 1880. Whereas all exemplar species of scorpions exhibit sperm packages, Buthida Soleglad and Fet, 2003, including Chaerilidae Pocock, 1893, and most Buthidae C.L. Koch, 1837, present unorganized sperm or loosely organized bundles. Although the details vary, three main types of sperm packages may be recognized in all other families: single folded; straight; and multiple folded. Subtypes may be identified according to general shape and folding patterns, mainly among sperm packages that are folded multiple times. Single-folded sperm packages are the most common type observed in the order. Although most sperm packages lack a covering, a conspicuous secretion sheath may be evident, e.g., in some Chactidae Pocock, 1893. Sperm packages vary in length from 112–354 µm and bent sperm packages are not necessarily longer than straight sperm packages. Four exemplar species of Bothriuridae reveal that variation in sperm count within a single sperm package is consistent with the count derived in spermatogenesis. The diversity of sperm packages suggests a path from free spermatozoa, via bent sperm packages, to other forms. Sperm packages may aid in the transport, cooperation, competition, and survival of spermatozoa. The diverse morphology, function, and evolution of sperm packages merit further investigation.

Sperm-cell DNA fragmentation prediction using label-free quantitative phase imaging and deep learning.

In intracytoplasmic sperm injection (ICSI), a single sperm cell is selected and injected into an egg. The quality of the chosen sperm and specifically its DNA fragmentation have a significant effect on the fertilization success rate. However, there is no method today to measure the DNA fragmentation of live and unstained cells during ICSI. We present a new method to predict the DNA fragmentation of sperm cells using multi-layer stain-free imaging data, including quantitative phase imaging, and lightweight deep learning architectures. The DNA fragmentation ground truth is achieved by staining the cells with acridine orange and imaging them via fluorescence microscopy. Our prediction model is based on the MobileNet convolutional neural network architecture combined with confidence measurement determined by distances between vectors in the latent space. Our results show that the mean absolute error for cells with high prediction confidence is 0.05 and the 90th percentile mean absolute error is 0.1, where the range of DNA fragmentation score is [0,1]. In the future, this model may be applied to improve cell selection by embryologists during ICSI.

Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Assisted Reproduction.

Remotely controllable microrobots are appealing for various biomedical in vivo applications. In particular, in recent years, ourgroup has focused on developing sperm-microcarriers to assist sperm cells with motion deficiencies or low sperm count (two of the most prominent male infertility problems) to reach the oocyte toward in-vivo-assisted fertilization. Different sperm carriers, considering their motion in realistic media and confined environments, have been optimized. However, the already-reported sperm carriers have been mainly designed to transport single sperm cell, with limited functionality. Thus, to take a step forward, here, the development of a 4D-printed multifunctional microcarrier containing soft and smart materials is reported. These microcarriers can not only transport and deliver multiple motile sperm cells, but also release heparin and mediate local enzymatic reactions by hyaluronidase-loaded polymersomes (HYAL-Psomes). These multifunctional facets enable in situ sperm capacitation/hyperactivation, and the local degradation of the cumulus complex that surrounds the oocyte, both to facilitate the sperm-oocyte interaction for the ultimate goal of in vivo assisted fertilization.

A novel solution for freezing individual spermatozoa using a right angular cryopiece embedded in a grooved petri dish.

Herein, we introduced a novel individual sperm freezing device named SpermCD, which consists of a right angular cryopiece (RA-Cryopiece, or "C") and a grooved petri dish ("D"). SpermCD allows embryologists to transfer sperm and perform ICSI on the same focal plane. Thirty-five patients underwent single sperm cryopreservation using SpermCD, including four patients with non-obstructive azoospermia (NOA), 14 patients with virtual azoospermia and 17 patients with cryptozoospermia. One hundred and twenty-five cryopreserved spermatozoa from nine patients were thawed on the day of the oocyte retrieval and 121 spermatozoa were found, with a sperm recovery rate of 97.1± 4.6%. Sixty-five MII oocytes from their spouse were injected with thawed sperm. Normal fertilization and high-quality embryo rates were 68.0% ± 33.2% and 24.4% ± 22.2%. Nineteen transplantable embryos were formed after fertilization with frozen sperm, eight of which were transplanted in five couples, resulting in four successful deliveries. SpermCD is a simple and practical individual sperm freezing device.

Separation of sperm and epithelial cells based on fluorescence-activated cell sorting

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.

Origin and timing of de novo variants implicated in type 2 von Willebrand disease.

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system-quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%-50% mutant cells) in three embryonic germ layer-derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.

SINGLE SPERM ATAC-SEQ REVEALS ALTERED CHROMATIN ACCESSIBILITY AT NEURODEVELOPMENTAL GENES IN ASSOCIATION WITH PATERNAL AGING

Paternal aging is associated with increased prevalence of childhood neurodevelopmental diseases, including autism spectrum disorder (ASD) and schizophrenia. During spermatogenesis, chromatin becomes tightly packaged around protamines, with <15% histones remaining at loci of embryonic developmental importance. We hypothesize that this critical process of chromatin reorganization is susceptible to perturbations as men age, leading to changes in sperm chromatin accessibility and epigenetic dysregulation. The aim of this study was to investigate at a single sperm level the variability of human sperm chromatin accessibility within an ejaculate as well as in association with advanced paternal age (APA).

Molecular detection of Vibrio paraheamolyticus in a sperm whale (Physeter macrocephalus) stranding in northern Veracruz, Mexico.

On 25 August 2021, a single female sperm whale (Physeter macrocephalus) was found stranded dead in Playa Tuxpan, Veracruz, Mexico. Skin biopsies were obtained and screened for the detection of various potentially pathogenic bacterial genera, using conventional polymerase chain reaction and sequencing of the positive amplicons. We recorded, for the first time, the presence of Vibrio paraheamolyticus in skin samples from P. macrocephalus in the Gulf of Mexico. Additionally, we discuss 29 records reporting strandings of sperm whales from six states of the Mexican Republic. Most of the records are concentrated in the Pacific Ocean. Our findings increase the inventory of bacteria reported in P. macrocephalus worldwide, summarising the knowledge of stranding events in sperm whale populations in Mexico.