Individual Human Sperm Research Articles

Chromosomal analysis of single sperm cells from infertile couples with severe oligoteratozoospermia: A cross-sectional prospective study.

In this cross-sectional prospective study, advanced next-generation sequencing technology was used to compare the molecular karyotyping of individual human sperm cells in infertile couples with severe oligoteratozoospermia (i.e., low sperm count and motility) to those of infertile couples with normal semen. Fourteen infertile couples who were patients at Ramathibodi Hospital in Bangkok, Thailand, were recruited from January to November 2023, and they were categorized into two groups based on semen analysis results. The study group comprised couples with severe oligoteratozoospermia, whereas the control group exhibited normal semen. Individual sperm cells from the semen samples were isolated by the micromanipulation technique for subsequent whole-genome amplification and next-generation sequencing, where the primary outcome was the aneuploidy rate. Seventy individual sperm cells were isolated with a 90% success rate for amplification. The next-generation sequencing results showed that the aneuploidy rate was 25%-75%, with a mean of 48.28% in the study group. In contrast, the control group exhibited aneuploidy rates of 0-75%, with a mean of 15.15%. The difference between the two groups was statistically significant (odds ratio: 5.8, 95% confidence interval: 1.30-26.03). Sperm cells of the study group showed a threefold higher aneuploidy rate than those in the control group, even though the sperm cells were selected by micromanipulation for their normal morphology. Comprehensive counseling is recommended to address elevated aneuploidy rates that potentially surpass those of the general infertile population. Guidance on preimplantation genetic testing is also recommended to ensure the transfer of embryos with normal chromosomes.

A method for low-coverage single-gamete sequence analysis demonstrates adherence to Mendel's first law across a large sample of human sperm.

Recently published single-cell sequencing data from individual human sperm (n=41,189; 969-3377 cells from each of 25 donors) offer an opportunity to investigate questions of inheritance with improved statistical power, but require new methods tailored to these extremely low-coverage data (∼0.01× per cell). To this end, we developed a method, named rhapsodi, that leverages sparse gamete genotype data to phase the diploid genomes of the donor individuals, impute missing gamete genotypes, and discover meiotic recombination breakpoints, benchmarking its performance across a wide range of study designs. We then applied rhapsodi to the sperm sequencing data to investigate adherence to Mendel's Law of Segregation, which states that the offspring of a diploid, heterozygous parent will inherit either allele with equal probability. While the vast majority of loci adhere to this rule, research in model and non-model organisms has uncovered numerous exceptions whereby 'selfish' alleles are disproportionately transmitted to the next generation. Evidence of such 'transmission distortion' (TD) in humans remains equivocal in part because scans of human pedigrees have been under-powered to detect small effects. After applying rhapsodi to the sperm data and scanning for evidence of TD, our results exhibited close concordance with binomial expectations under balanced transmission. Together, our work demonstrates that rhapsodi can facilitate novel uses of inferred genotype data and meiotic recombination events, while offering a powerful quantitative framework for testing for TD in other cohorts and study systems.

High-resolution 4-D acquisition of freely swimming human sperm cells without staining.

We present a new acquisition method that enables high-resolution, fine-detail full reconstruction of the three-dimensional movement and structure of individual human sperm cells swimming freely. We achieve both retrieval of the three-dimensional refractive-index profile of the sperm head, revealing its fine internal organelles and time-varying orientation, and the detailed four-dimensional localization of the thin, highly-dynamic flagellum of the sperm cell. Live human sperm cells were acquired during free swim using a high-speed off-axis holographic system that does not require any moving elements or cell staining. The reconstruction is based solely on the natural movement of the sperm cell and a novel set of algorithms, enabling the detailed four-dimensional recovery. Using this refractive-index imaging approach, we believe that we have detected an area in the cell that is attributed to the centriole. This method has great potential for both biological assays and clinical use of intact sperm cells.

The application of Raman spectroscopy in male reproduction diagnosis

The limited method demands for new technology in the management of male infertility. Raman spectroscopy is a rapid and sensitive non-invasive diagnosing technology. Raman spectroscopy was able to obtain Raman spectra from individual human sperm cells and allows for the comparison of their biochemical components, DNA and mitochondrial structure as well as epigenetic changes. This review provided an overview of the principle of each major types and discussed the progress in clinical application, indicating the potential of Raman spectroscopy as an valuable addition in assessing the quality of sperm cells for in vitro fertilization. Key words: Infertility, male; Spermatozoa; Semen; Spectrum analysis, Raman

Continuous behavioural 'switching' in human spermatozoa and its regulation by Ca2+-mobilising stimuli.

Human sperm show a variety of different behaviours (types of motility) that have different functional roles. Previous reports suggest that sperm may reversibly switch between these behaviours. We have recorded and analysed the behaviour of individual human sperm (180 cells in total), each cell monitored continuously for 3–3.5 min either under control conditions or in the presence of Ca2+-mobilising stimuli. Switching between different behaviours was assessed visually (1 s bins using four behaviour categories), and was verified by fractal dimension analysis of sperm head tracks. In the absence of stimuli, ~90% of cells showed at least one behavioural transition (mean rate under control conditions = 6.4 ± 0.8 transitions.min−1). Type 1 behaviour (progressive, activated-like motility) was most common, but the majority of cells (>70%) displayed at least three behaviour types. Treatment of sperm with Ca2+-mobilising agonists had negligible effects on the rate of switching but increased the time spent in type 2 and type 3 (hyperactivation-like) behaviours (P < 2*10−8; chi-square). Treatment with 4-aminopyridine under alkaline conditions (pHo = 8.5), a highly-potent Ca2+-mobilising stimulus, was the most effective in increasing the proportion of type 3 behaviour, biasing switching away from type 1 (P < 0.005) and dramatically extending the duration of type 3 events (P < 10−16). Other stimuli, including 300 nM progesterone and 1% human follicular fluid, had qualitatively similar effects but were less potent. We conclude that human sperm observed in vitro constitutively display a range of behaviours and regulation of motility by [Ca2+]i, at the level of the single cell, is achieved not by causing cells to adopt a ‘new’ behaviour but by changing the relative contributions of those behaviours.

Protamine 2 Precursors and Processing

Protamine 2 (P2) is synthesised as a precursor protein (pre-P2) which by proteolysis is processed to generate the mature components of the protamine 2 family of proteins (HP2, HP3 and HP4). In infertile patients, abnormal processing of the protamine 2 precursors has been suggested by the detection of an increased presence of precursor forms. However, the presence of small detectable amounts of precursor proteins has been demonstrated also in normal sperm samples, although the variation of pre-P2 in individual human sperm cells had not yet been explored. In the present manuscript we perform a mini-review describing what is known about protamine 2 precursors and P2 processing. In addition, we by immunofluorescence demonstrate the existence of a marked variation in the presence and abundance of pre-P2 in individual sperm cells.

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

Atomic Force Microscope Analysis of Chromatin Volumes in Human Sperm with Head-Shape Abnormalities1

We used the atomic force microscope to perform volume measurements on individual human sperm nuclei under ambient conditions. Data obtained for normal sperm and for sperm from seven of the nine classes of head-shape abnormality revealed that the nuclear volumes were essentially identical for sperm in each headshape class, even though the projected areas and shapes of the nuclei have were shown to vary widely. These results indicate that the abnormal sperm head morphologies found at a rate of 25-40% in fertile males are not caused by factors that affect the volume of sperm chromatin, such as the DNA content of the sperm nucleus, differences in chromatin organization, or the extent of DNA compaction.

Characteristics of Calcium Ion Influx Induced by Human Follicular Fluid in Individual Human Sperm

The mechanism of Ca2+ influx into human spermatozoa was investigated. Human follicular fluid (hFF), which contains progesterone, stimulated an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) as measured by fluorescence spectroscopy using Fura-2/AM. The technique allowed the measurement of [Ca2+]i in individual, motile spermatozoa, by improvement of the previous methods. The role of Ca2+ channels was investigated using selective blocking agents. Neither blockade of L-type channels with verapamil or lanthanum (La3+) nor blockade of N-type channels with omega-conotoxin affected the hFF-stimulated increase in [Ca2+]i. Blockade of T-type channels with either nickel (Ni2+) or amiloride significantly reduced the hFF-stimulated increase in [Ca2+]i and amiloride significantly decreased baseline [Ca2+]i. Incubation of sperm in Na(+)-free medium did not affect [Ca2+]i, indicating that Na+/Ca2+ exchange has no major role in Ca2+ influx. The results suggest that calcium influx induced by hFF occurs via T-type voltage-independent Ca2+ channels. The method described allows accurate measurement of [Ca2+]i in individual, motile human spermatozoa.

Intracellular calcium measurements in individual human sperm demonstrate that the majority can respond to progesterone

To determine if human spermatozoa could be immobilized and intracellular calcium measurements made on individual cells to measure what proportion can respond to P. Spermatozoa were loaded with Fura 2 (Sigma Chemical Co., Poole, Dorest, United Kingdom) and suspended in 10% gelatin at 37 degrees C. A thin layer of the suspension was cooled to room temperature (20 degrees C to 25 degrees C) and [Ca2+]i was measured with a fluorescence microscope equipped with dual wavelength excitation and an image analysis system. University-based laboratory. Semen was obtained from four fertile donors to a donor insemination program. None. [Ca2+]i was calculated from the ratio of Fura 2 fluorescence excited at 340 nm and that excited at 366 nm. One hundred six of 114 sperm examined (93%) demonstrated a significant response to P but the size and duration of the response was variable. These data demonstrate that most sperm can respond to P.

Increase of intracellular free [Ca2+] in single human motile spermatozoa treated with human follicular fluid.

The intracellular free [Ca2+] concentration ([Ca2+]i) in individual human sperm was measured using a fluorescent Ca2+ indicator. In 18 of 23 motile sperm (78.3%), [Ca2+]i increased significantly and promptly after addition of 20% human follicular fluid (hFF), but in the others it did not increase. The mean resting [Ca2+]i level of sperm in which [Ca2+]i increased after addition of 20% hFF (the influx group) was significantly lower than those in which it did not increase (112.8 +/- 40.1 nM vs. 156.9 +/- 13.5 nM, p < .05). After addition of 20% hFF, the mean [Ca2+]i in the influx group reached a peak value of 210.7 +/- 24.7 nM within 30 s and then decreased slowly; the mean [Ca2+]i values 1, 5, 10, and 15 min after addition of 20% hFF were 179.3 +/- 31.4, 174.3 +/- 30.2, 172.5 +/- 27.8, and 175.1 +/- 27.2 nM, and all values were significantly higher than the resting level (p < .01). The frequency distribution of [Ca2+]i after addition of 20% hFF was shifted toward higher concentrations (p < .01). However, the addition of 20% hFF did not increase the percentage of live acrosome reaction (before 3.8 +/- 0.9% vs. after 2.9 +/- 0.5%, respectively). Thus, hFF increased [Ca2+]i in about 80% of the motile sperm. Relatively high [Ca2+]i levels persisted for at least 10-15 min after its addition. However, hFF did not trigger a rapid response in acrosome reaction.

Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa.

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.

Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromatic region on the long arm of the human Y chromosome and for unique DNA sequences on human chromosome 19. Chromosome 4 occupied a circumscribed domain in the pronuclei, similar to findings in somatic interphases. Unlike the situation in somatic interphases, the Y heterochromatin was extended throughout the first cell cycle. Pronuclear chromatin was extended 3- to 4-fold compared to somatic interphase chromatin. The extended pronuclear chromatin conformation is likely to affect a zygote's susceptibility to environmental hazards.

Controlled micromanipulation of human sperm in three dimensions with an infrared laser optical trap: effect on sperm velocity

Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG laser trap. Single sperm swimming with velocities in the range of 65 to 85 microns/sec can be trapped with 40 mW of power through 120 seconds without a deleterious effect on velocity. Even though it will be necessary to further evaluate the effects of laser light on specific functions of sperm, our data suggest that decreasing the time of manipulation to a minimum will increase the safety of the micromanipulation procedures. Laser traps may play a role in assisted reproductive technology by facilitating the selective transport of individual sperm.

Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus

It is well known that cervical mucus restricts penetration of morphologically abnormal human sperm, both in vitro and in vivo. However, the mechanisms of such restriction are not well understood. Using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, we analyzed differential penetration of normal and abnormal sperm into fresh human cervical mucus. Abnormal sperm swam slower in mucus than the normal sperm, but their flagellar beat parameters were not commensurately different. Multivariate statistical analysis of the relationship between individual sperm velocity and flagellar beat parameters indicated that the heads of the abnormal sperm experienced greater resistance from the mucus than did normal heads. Differential mucus resistance, more than altered motile vigor, appears to be responsible for the restriction of abnormal sperm during migration through mucus.

Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus

It is well known that cervical mucus restricts penetration of morphologically abnormal human sperm, both in vitro and in vivo. However, the mechanisms of such restriction are not well understood. Using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, we analyzed differential penetration of normal and abnormal sperm into fresh human cervical mucus. Abnormal sperm swam slower in mucus than the normal sperm, but their flagellar beat parameters were not commensurately different. Multivariate statistical analysis of the relationship between individual sperm velocity and flagellar beat parameters indicated that the heads of the abnormal sperm experienced greater resistance from the mucus than did normal heads. Differential mucus resistance, more than altered motile vigor, appears to be responsible for the restriction of abnormal sperm during migration through mucus.

Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.

Spontaneous lipid peroxidation in washed human spermatozoa was induced by aerobic incubation at 32 C and measured by malonaldehyde production; loss of motility during the incubation was determined simultaneously. Malonaldehyde production at the point of complete loss of motility, defined as the lipoperoxidative lethal endpoint (LLE), was 0.10 +/- 0.03 nmol/10(8) cells (mean +/- SD, n = 40), and was independent of the time to complete loss of motility. Human spermatozoa produced both H2O2 and O2-. during aerobic incubation. Inhibition of superoxide dismutase in these cells with KCN showed that all the H2O2 production is due to action of the dismutase. The superoxide dismutase activity of individual human sperm samples varied between 1 and 10 U/10(8) cells, variations between samples from a single donor being nearly as great as those between different donors. The time to complete motility loss (tL) showed equal variation of 1 to 10 hours among samples. The rate of spontaneous lipid peroxidation, calculated as LLE/tL, for a given sperm sample and the superoxide dismutase activity of the same sample, determined prior to aerobic incubation, gave a good linear correlation (r = 0.97). Glutathione reductase, glutathione peroxidase, and glutathione were found to be present in human spermatozoa, but showed little variation among samples. These results suggest that superoxide dismutase plays the major role in protecting human spermatozoa against lipid peroxidation. In addition, the superoxide dismutase activity of a fresh sperm sample appears to be a good predictor of the lifetime (up to the complete loss of motility) of that particular sample, and so may prove useful in semen analysis.