Uses and methods for elemental characterization in the diagnosis and prognosis of medical diseases and conditions - Patents.com

Abstract

The present application provides a method for detecting infertile sperm in a sample, the method comprising detecting a concentration of at least one metal in the sample that is outside a predetermined range using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS). It also provides a method for detecting infertile sperm in a sample, the method comprising detecting a dynamic or kinetic parameter of a signal spike of at least one metal selected from a group of several metal elements by sc-ICP-MS.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to Chinese Patent Application No. 202110493989.4, filed on May 7, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present technology relates to the field of medical diagnostics, patient monitoring, and treatment efficacy evaluation. It is directed to methods, uses, and kits related to detecting (i) the concentration, or (ii) the dynamic or kinetic parameters of the signal spike, or both (i) and (ii), of at least one metal in sperm using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS). The technology is suitable for detecting fertile or infertile sperm in a sample. [Background technology]

[0003] The following explanation is provided to aid the reader's understanding. None of the information provided or references cited are admitted to be prior art.

[0004] Infertility has become a common health problem and was identified as a disease by the World Health Organization (WHO) in 2009 (see, e.g., Zegers-Hochschild et al., Fertil Steril, 2009, 92, 1520-1524), just third after cancer and cardiovascular disease. Currently, infertility affects approximately 15% of reproductive-age couples worldwide (see, e.g., Levine et al., Hum Reprod Update, 2017, 23, 646-659, and Joffe, Human Reproduction, 2010, 25, 295-307). Male factors account for approximately 50% of all infertility cases and in some clinics may account for up to 70% of male evaluations (see, e.g., Agarwal et al., Reprod Biol Endocrinol, 2015, 13, 37, and Tuttelmann et al., Med Genet, 2018, 30, 12-20).

[0005] The main factors causing male infertility are sperm dysfunction, sperm oliguria, infections, sexual dysfunction, and endocrine and genetic disorders (Krausz and Riera-Escamilla, 2018, Nat Rev Urol 15, 369-384; Mehra et al., 2018, Urologia 85, 22-24; Punab et al., 2017, Hum Reprod 32, 18-31; Vander Borght and Wyns, 2018, Clin Biochem 62, 2-10; Wall and Jayasena, 2018, BMJ 363, k3202; and Zhou et al., 2018, BJOG 125, 432-441). Among these factors, azoospermia, asthenozoospermia, sexual dysfunction, and other symptoms related to sperm physical parameters are easier to diagnose with traditional methods (Centola, 2014, Urol Clin N Am 41, 163-7; Krausz and Riera-Escamilla, Punab et al., Wall and Jayasena, and WHO, WHO laboratory manual for the examination and processing of human semen, World Health Organization, Geneva, Switzerland, 6th edn., 2021). Currently, clinical analysis of male infertility mainly relies on semen analysis and body-based sperm parameters, and traditional male infertility diagnostic methods mainly focus on sperm morphological observation and seminal plasma biochemical component analysis, such as sperm morphology and motility, semen appearance, immunological evaluation, seminal plasma biochemical detection, and karyotype parameters, according to the WHO Laboratory Manual for Examination and Processing of Human Semen, 6th edition (see WHO, 2021).However, there are still approximately 30% of infertile men diagnosed with normal semen analysis, diagnostic failure rates still range from 30% to 70% of clinical cases, and most of the cases, up to 72% of male infertility cases in some developed countries, are still classified as idiopathic (see, e.g., Agarwal et al., 2021, Lancet 397, 319-333; Hamada et al., 2012, Int Braz J Urol 38, 576-594; Mascarenhas et al., 2012. Plos Med 9; and Tuttelmann et al.).

[0006] Thus, there is an urgent need for new techniques for assessing the functional quality of human sperm. The present disclosure fulfills this need and also provides related advantages. Summary of the Invention

[0007] In one aspect, the disclosure provides a method for detecting infertile sperm in a sample obtained from a subject.

[0008] In some embodiments, the method comprises, consists essentially of, or even consists of detecting a concentration of at least one metal in a sample that is outside a predetermined range using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS).

[0009] In some embodiments, the at least one metal is selected from the group consisting of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni).

[0010] In some embodiments, the sample is diluted with a buffer prior to the detecting step.

[0011] In some embodiments, the predetermined range corresponds to a concentration of the metal detected in sperm from a population of fertile subjects.

[0012] In some embodiments, the sperm are capacitated.

[0013] In some embodiments, the sperm are not capacitated.

[0014] In some embodiments, the predetermined range for Na concentration is about 5 attograms (ag) to about 50,000 ag for uncapacitated sperm and about 25 ag to about 50,000 ag for capacitated sperm; the predetermined range for K concentration is about 50 ag to about 50,000 ag for uncapacitated sperm and about 280 ag to about 50,000 ag for capacitated sperm; and the predetermined range for Ca concentration is about 200 ag to about 50,000 ag for uncapacitated sperm. For capacitated sperm, the predetermined range is about 700ag to about 20,500ag; for Mg concentration, the predetermined range is about 8ag to about 50,000ag for uncapacitated sperm and about 75ag to about 15,100ag for capacitated sperm; for Zn concentration, the predetermined range is about 5ag to about 50,000ag for uncapacitated sperm and about 20ag to about 50,000ag for capacitated sperm; and for Fe concentration, the predetermined range is about 5ag to about 50,000ag for uncapacitated sperm. for capacitated sperm, the predetermined range of Al concentration is about 3 ag to about 50,000 ag for uncapacitated sperm, and about 6 ag to about 50,000 ag for capacitated sperm (e.g., about 6 ag to about 46,700 ag, etc.); for Se concentration, the predetermined range is about 59 ag to about 50,000 ag for uncapacitated sperm, and about 62 ag to about 45,810 ag for capacitated sperm; The predetermined range of O concentration is about 3 ag to about 3,700 ag for uncapacitated sperm and about 9 ag to about 20,200 ag for capacitated sperm, the predetermined range of Cu concentration is about 9 ng to about 50,000 ag for uncapacitated sperm and about 9 ng to about 37,590 ag for capacitated sperm, and the predetermined range of Cr concentration is about 4 ng to about 50,000 ag for uncapacitated sperm and about 5 ag to about 46,000 ag for capacitated sperm.000 ag, and the predetermined range of Mn concentration is from about 2 ag to about 50,000 ag for uncapacitated sperm and from about 7 ag to about 32,610 ag for capacitated sperm.

[0015] In one aspect, the present disclosure provides a method for detecting infertile sperm in a sample obtained from a subject, comprising detecting a dynamic or kinetic parameter of a signal spike of at least one metal selected from the group of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni) in the sample that is outside a predetermined range by single cell inductively coupled plasma mass spectrometry (sc-ICP-MS).

[0016] In some embodiments, the parameters correspond to dynamic or kinetic parameters detected in sperm from a population of fertile subjects.

[0017] In some embodiments, the dynamic or kinetic parameters of the spike are selected from dwell time, dwell time before the peak, dwell time after the peak, peak time, ratio between peak time and dwell time, rising tau constant before the peak, dynamic area before the peak, tailing tau constant after the peak, dynamic area after the peak, or any combination thereof.

[0018] In some embodiments, the dynamic or kinetic parameters of the spike include: (a) a dwell time of the Fe spike of about 1.4 to about 7.9 ms for uncapacitated sperm, and a dwell time of the Fe spike of about 1.5 to about 6.7 ms for capacitated sperm; (b) a tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.81 ms for uncapacitated sperm, and a tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.90 ms for capacitated sperm; (c) a tailing tau constant after the peak of the Fe spike of about 0.20 to about 0.25 ms for capacitated sperm; (d) a dwell time of the Cu spike of approximately 1.5 ms or less for uncapacitated sperm and capacitated sperm; (e) a dwell time of the Cu spike of approximately -0.2 ms or less for uncapacitated sperm and approximately -0.6 ms or less for capacitated sperm. (f) a tailing tau constant after the peak of the Cu spike of about 0.15 ms or less for uncapacitated sperm, and a tailing tau constant after the peak of the Cu spike of about 0.2 ms or less for capacitated sperm; (g) a dwell time of the Zn spike of about 2.1 ms or less for uncapacitated sperm, and a dwell time of the Zn spike of about 1.2 ms or less for capacitated sperm; (h) a dwell time of the Zn spike of about -0.25 ms or less for uncapacitated sperm. (i) a rising tau constant before the peak of the Zn spike less than or equal to about -0.20 ms for capacitated sperm, (ii) a tailing tau constant after the peak of the Zn spike less than or equal to about 1.15 ms for uncapacitated sperm, and (iii) a tailing tau constant after the peak of the Zn spike less than or equal to about 0.25 ms for capacitated sperm, (j) a residence time of the Cr spike less than or equal to about 3.25 ms for uncapacitated sperm, and (k) a tailing tau constant after the peak of the Zn spike less than or equal to about -0.(k) for uncapacitated sperm, a tailing tau constant after the peak of the Cr spike of about 0.2 to about 0.5 ms; (l) for uncapacitated sperm, a residence time of the Se spike of about 1.5 ms or less; (m) for uncapacitated sperm, a rising tau constant before the peak of the Se spike of about -0.15 ms or less; and (n) for uncapacitated sperm, a tailing tau constant after the peak of the Se spike of about 0.25 ms or less.

[0019] In certain aspects, the disclosure provides a method for detecting infertile sperm in a sample, comprising: (i) contacting a first population of sperm from a subject with a human tubal fluid (HTF) buffer; and (ii) using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) to detect a concentration of at least one metal selected from the group of potassium (K), calcium (Ca), magnesium (Mg), mercury (Hg), silver (Ag), and aluminum (Al) in the first population of sperm after the contacting step that is equal to or lower than the concentration present in a second population of sperm that has not been contacted with the HTF buffer; or (iii) using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) to detect a concentration of at least one metal selected from the group of potassium (K), calcium (Ca), magnesium (Mg), mercury (Hg), silver (Ag), and aluminum (Al) in the first population of sperm after the contacting step that is equal to or lower than the concentration present in a second population of sperm that has not been contacted with the HTF buffer. or (iv) detecting a concentration of selenium (Se) in the first population of sperm after the contacting step that is equal to or greater than the concentration present in a second population of sperm that has not been contacted with the HTF buffer using sc-ICP-MS; or (v) detecting a concentration of selenium (Se) in the first population of sperm after the contacting step that is equal to or greater than the concentration present in a second population of sperm that has not been contacted with the HTF buffer using sc-ICP-MS.

[0020] In some embodiments, the sample is obtained from a subject.

[0021] In some embodiments, the subject has or is suspected of having idiopathic infertility, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia.

[0022] In some embodiments, the method further comprises treating the subject with an infertility therapy or treatment.

[0023] In some embodiments, the concentration of at least one metal in the sperm is below a predetermined range and the infertility therapy comprises treatment with the at least one metal.

[0024] In some embodiments, more than one of the metals is detected.

[0025] In some embodiments, the sperm are at least 3×10 6 The sperm are diluted to a concentration of less than 100 sperm / ml.

[0026] In some embodiments, the sperm are diluted 10-fold or more prior to the detecting step.

[0027] In some embodiments, the sperm are centrifuged to remove seminal plasma before dilution.

[0028] In some embodiments the sample comprises semen that is optionally liquefied, or fixed, or capacitated, or cryopreserved, or liquefied and fixed, or liquefied and capacitated, or liquefied and cryopreserved, or fixed and capacitated, or fixed and cryopreserved, or capacitated and cryopreserved, or liquefied, fixed and capacitated, or liquefied, fixed and cryopreserved, or liquefied, capacitated and cryopreserved, or liquefied, fixed, capacitated and cryopreserved.

[0029] In some embodiments, the method further comprises performing computer-aided sperm analysis (CASA) on the sperm.

[0030] In some embodiments, the method further comprises purifying sperm having (i) at least one metal concentration, or (ii) a dynamic or kinetic parameter, or both (i) and (ii), within a predetermined range to obtain functional sperm.

[0031] In some embodiments, the method optionally further comprises fertilizing an egg with the purified functional sperm via in vivo fertilization (IVF) or intracellular sperm injection (ICSI).

[0032] In one aspect, the present disclosure provides kits for practicing the methods disclosed herein.

[0033] In some embodiments, the kit comprises a buffer and instructions for practicing the methods disclosed herein. [Brief description of the drawings]

[0034] [Figure 1] Figure 1 shows the quantitative analysis of multiple elements in single human sperm in different samples.By using the method disclosed herein, it is possible to detect the difference of multiple elements in different samples.The lowest sensitivity range for the detection of multiple elements can reach single cell level with a concentration of 10 billionth of a gram (ag). [Diagram 2] Using the method disclosed herein, examples of samples with relatively high content of essential trace elements are shown to be detected in single human sperm. These findings can be used as reference standards, and suggestions for supplementary elements can be made to people with deficiencies of these essential elements. [Diagram 3]Provide an example of a sample with relatively high content of toxic elements in a single human sperm. Clinical detoxification treatment can be proposed for those samples of subjects with relatively high levels of toxic elements. [Figure 4] An example of a sample with a relatively high content of elements such as nickel (Ni) in a single human sperm is shown. [Diagram 5] Provides unique characteristics of ICP-MS signal spikes for specific elements iron (Fe) and copper (Cu) in single human sperm. The sc-ICP-MS signal kinetics of iron (Fe) and copper (Cu) elements are detected in the same batch of single human sperm cells in A, B, and C of the same sample. The signal kinetics includes the total peak dwell time, the peak time of the spike signal, the dwell time before or after the peak, the dynamic rising tau constant and area before the peak, and the dynamic tailing tau constant and area after the peak. These parameters are characteristic of the specific elemental signal of a given cell type with a unique elemental profile. [Figure 6] Provides signaling characteristics of essential macronutrients (calcium and magnesium) in different cell types. [Figure 7] It provides signal characteristics of essential trace elements in single human sperm, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), cobalt (Co), selenium (Se), etc. [Figure 8] To provide the signaling characteristics of essential trace elements in mouse epididymal epithelial DC2 cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), cobalt (Co), and selenium (Se). [Figure 9] To provide the signaling characteristics of essential trace elements in human embryonic kidney 293T cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), and selenium (Se). [Figure 10] To provide the signal characteristics of essential trace elements in human cervical cancer HeLa cells, including zinc (Zn), iron (Fe), manganese (Mn), and chromium (Cr). [Figure 11]To provide the signal characteristics of essential trace elements in human gastric cancer SNU-1 cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and chromium (Cr). [Figure 12A] The signal characteristics of toxic elements in different cell types, including human sperm from semen, are provided. The biotoxicological and pathological significance of the signal values ​​of these toxic elements and their kinetic characteristics, as well as the prospects for clinical diagnosis, are being further investigated. [Figure 12B] The signal characteristics of toxic elements in different cell types, including cultured mouse epididymal epithelial DC2 cells, are presented. The biotoxicological and pathological significance of the signal values ​​of these toxic elements and their kinetic characteristics, as well as the prospects for clinical diagnosis, are under further investigation. [Figure 12C] The signal characteristics of toxic elements in different cell types, including cultured human embryonic kidney 293T cells, are provided. The biotoxicological and pathological significance of the signal values ​​of these toxic elements and their kinetic characteristics, as well as the prospects for clinical diagnosis, are under further investigation. [Figure 12D] The signal characteristics of toxic elements in different cell types, including cultured human cervical cancer HeLa cells, are provided. The biotoxicological and pathological significance of the signal values ​​of these toxic elements and their kinetic characteristics, as well as the prospects for clinical diagnosis, are under further investigation. [Figure 12E] The signal characteristics of toxic elements in different cell types, including cultured human gastric cancer SNU-1 cells, are provided. The biotoxicological and pathological significance of the signal values ​​of these toxic elements and their kinetic characteristics, as well as the prospects for clinical diagnosis, are under further investigation. [Figure 13A]The mean masses of elements in single normal human sperm are provided. The mean masses of elements detected in single human sperm from all populations in samples with normal (represented as original) sperm density lower or higher than 1×106 per ml, or samples capacitated with viable DGCs (represented as viable DGCs) with a density of 1×106 per ml or less are shown. Data are expressed as mean ± SEM (n=3-30 samples), 3-way ANOVA, *P<0.05, ****P<0.0001. ND: not determined due to being outside the detection range. [Figure 13B] The mean mass of elements in single normal human sperm is provided. The mean mass of elements detected in single human sperm from the entire population in normal samples (represented as Original(Low)) or oligoasthenozoospermia sperm samples (represented as Oligoastheno), both of which have a density of 1×106 per ml or less, are shown. Data are expressed as mean ± SEM (n=3-30 samples), 3-way ANOVA, *P<0.05, ****P<0.0001. ND: Not determined due to being outside the detection range. [Figure 13C] The mean mass of elements in single normal human sperm is provided. The fold change in mean mass of elements in samples with high versus low sperm density is shown. Data are expressed as mean ± SEM (n = 3-30 samples), 3-way ANOVA, *P < 0.05, ****P < 0.0001. ND: Not determined due to being outside the detection range. [Figure 13D] The mean mass of elements in single normal human sperm is provided. The fold change in mean mass of elements in capacitated DGC stimulated samples versus non-capacitated samples of either normal sperm cells or oligoasthenozoospermia sperm cells is shown. Data are expressed as mean ± SEM (n = 3-30 samples), 3-way ANOVA, *P < 0.05, ****P < 0.0001. ND: Not determined due to being outside the detection range. [Figure 14A] FIG. 1 provides the correlation of the average mass of K vs. Na in human sperm of high or low density, normal or oligoastheno. samples. The arrows indicate the inverse correlation of K and Na. [Figure 14B]A comparison of the mean masses of elements detected in human sperm using sc-ICP-MS and conventional digestion ICP-MS measurements is provided. The mean masses of elements detected in single human sperm from each population of normal or viable DGC samples with a density of 1 x 106 per ml or less, or control normozoospermia or oligoasthenospermia samples, are shown. Data are expressed as mean ± SEM (n = 3-30 samples), 3-way ANOVA, ****P < 0.0001. ND: not determined due to being outside the detection range. [Figure 14C] A comparison of the average masses of elements detected in human sperm using sc-ICP-MS and conventional digested ICP-MS measurements is provided. The average masses of elements detected in a batch of digested human sperm samples as in FIG. 14B are shown. Data are expressed as mean ± SEM (n = 3-30 samples), 3-way ANOVA, ****P < 0.0001. ND: Not determined due to being outside the detection range. [Figure 15] Comparisons of the mean masses of elements in single human sperm (with a density of less than 1 x 106 per ml) as well as other somatic cell cultures, including mouse WT epididymal epithelial DC2 cells, human embryonic kidney 293T cells, human cervical cancer HeLa cells, and human gastric cancer SNU-1 cells, are provided. Data are expressed as mean ± SEM (n = 3-30 samples), 2-way ANOVA, *P < 0.05, ****P < 0.0001. ND: Not determined due to being outside the detection range. [Figure 16A]Correlation of the mean mass of elements to Ca in single human sperm from normal and oligoasthenospermic subjects is provided. Mean mass of essential macroelements (Na, K and Mg) per sperm to mean mass of Ca in single human sperm from samples prepared from original semen (represented as original) or normal viable DGC-capacitated sperm in human tubal fluid (HTF) solution. Data points were obtained from an average of at least three cells per sample at a cell density of less than 1 x 106 per ml of normal or oligoasthenospermic sperm diluted from original semen or after DGC-capacitation treatment. Arrows indicate the inverse correlation potential between elements and Ca. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 16B] Correlation of the mean mass of elements to Ca in single human sperm from normal and oligoasthenospermic subjects is provided. Mean mass of essential trace elements (Co, Cr, Cu, Fe, Mn, Se and Zn) per sperm to mean mass of Ca in single human sperm from samples prepared from original semen (represented as original) or normal viable DGC-capacitated sperm in human tubal fluid (HTF) solution. Data points were obtained from an average of at least three cells per sample at a cell density of less than 1×106 per ml of normal or oligoasthenospermic sperm diluted from original semen or after DGC-capacitation treatment. Arrows indicate the inverse correlation potential between elements and Ca. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 16C]Correlation of the mean mass of elements to Ca in single human sperm from normal and oligoasthenospermic subjects is provided. The mean mass of other or toxic elements (As, Al, Ag, Cd, Hg, Ni and Pb) per sperm to the mean mass of Ca in single human sperm from samples prepared from original semen (represented as original) or normal viable DGC-capacitated sperm in human tubal fluid (HTF) solution. Data points were obtained from the average of at least three cells per sample at a cell density of less than 1 x 106 per ml of normal or oligoasthenospermic sperm diluted from original semen or after DGC-capacitation treatment. Arrows indicate the inverse correlation potential between elements and Ca. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 17A] Correlation of the mean mass of elements to Ca in single human sperm at high or low cell density is provided. Mean mass of essential macroelements (Na, K and Mg) per sperm to mean mass of Ca in single human sperm from samples prepared from original semen (designated as original) or normal viable DGC-capacitated sperm in HTF solution. Data points were obtained from an average of at least three cells per sample at higher or lower cell densities than 1 x 106 per ml of normal or oligoasthenozoospermic sperm diluted from original semen or after DGC-capacitation treatment. Lines indicate data obtained from the same sample. [Figure 17B] Correlation of the mean mass of elements to Ca in single human sperm at high or low cell density is provided. Mean mass of essential trace elements (Co, Cr, Cu, Fe, Mn, Se and Zn) per sperm to mean mass of Ca in single human sperm of samples prepared from original semen (represented as original) or normal viable DGC-capacitated sperm in HTF solution. Data points were obtained from an average of at least three cells per sample at higher or lower cell densities than 1 x 106 per ml of normal or oligoasthenozoospermic sperm diluted from original semen or after DGC-capacitation treatment. Lines indicate data obtained from the same sample. [Figure 17C] Correlation of the mean mass of elements to Ca in single human sperm at high or low cell density is provided. The mean mass of other or toxic elements (As, Al, Ag, Cd, Hg, Ni and Pb) per sperm to the mean mass of Ca in single human sperm of samples prepared from original semen (represented as original) or normal viable DGC-capacitated sperm in HTF solution. Data points were obtained from an average of at least three cells per sample at higher or lower cell densities than 1 x 106 per ml of normal or oligoasthenozoospermic sperm diluted from original semen or after DGC-capacitation treatment. Lines indicate data obtained from the same sample. [Figure 18A] 1 provides correlation of the average mass of elements to Ca in single mouse epididymal epithelial DC2 cells. Average mass correlation of essential macroelements (Na, K and Mg) to Ca from a single ICP-MS signal spike of an aspirated single cell. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 18B] Figure 1 provides correlation of average mass of elements to Ca in single mouse epididymal epithelial DC2 cells. Average mass correlation of essential trace elements (Co, Cr, Cu, Fe, Mn, Se and Zn) to Ca from single ICP-MS signal spikes of aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 18C] Provides correlation of average mass of elements to Ca in single mouse epididymal epithelial DC2 cells. Average mass correlation of other or toxic elements (As, Al, Ag, Hg, Ni and Pb) to Ca by single ICP-MS signal spike of aspirated single cells. Data points were obtained from the average of at least three cells per sample. Lines indicate data obtained from the same sample. ND: Not determined due to being outside the detection range. [Figure 19A]1 provides a correlation of the average mass of elements to Ca in human embryonic kidney HEK293T cells. The average mass of essential macroelements (Na, K, and Mg) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 19B] FIG. 1 provides a correlation of the average mass of elements to Ca in human embryonic kidney HEK293T cells. The average mass of essential trace elements (Co, Cr, Cu, Fe, Mn, Se, and Zn) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 19C] 1 provides a correlation of the average mass of elements to Ca in human embryonic kidney HEK293T cells. The average mass of other or toxic elements (As, Al, Ag, Hg, Ni, and Pb) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 20A] 1 provides a correlation of the average mass of elements to Ca in human cervical cancer HELA cells. The average mass of essential macroelements (Na, K and Mg) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 20B] Correlation of the average mass of elements to Ca in human cervical cancer HELA cells. Average mass of essential trace elements (Co, Cr, Cu, Fe, Mn, Se and Zn) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 20C]Correlation of the average mass of elements to Ca in human cervical cancer HELA cells. The average mass of other or toxic elements (As, Al, Ag, Hg, Ni and Pb) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 21A] 1 provides a correlation of the average mass of elements to Ca in human gastric cancer SNU-1 cells. The average mass of essential macroelements (Na, K and Mg) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 21B] Correlation of the average mass of elements to Ca in human gastric cancer SNU-1 cells. Average mass of essential trace elements (Co, Cr, Cu, Fe, Mn, Se and Zn) per cell to Ca in aspirated single cells. Data points were obtained from an average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 21C] Correlation of the average mass of elements to Ca in human gastric cancer SNU-1 cells. The average mass of other or toxic elements (As, Al, Ag, Hg, Ni, and Pb) per cell to Ca in aspirated single cells. Data points were obtained from the average of at least three cells per sample. Lines indicate data obtained from the same sample. [Figure 22]Provides frequency distribution characteristics of the mean mass of various elements detected in single human sperm. Normal or oligoasthenospermia samples (represented as normal / original or abnormal / original) prepared directly from normal original semen for viable sperm treated with standard procedure of density gradient centrifugation in HTF solution (represented as normal / capacitation) for sperm undergoing capacitation during centrifugation (represented as abnormal / capacitation). ND: Not determined due to being outside the detection range. Data were detected at cell densities of less than 1 x 106 human sperm per ml in single human sperm and expressed as mean ± SEM (n = 3-30 individual samples), 2-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. [Figure 23A] We provide an association between elementomic mean mass frequency patterns and oligoasthenospermia risk. Using Fe elemental analysis as an example, we show that detailed frequency distribution analysis of the mean mass of Fe detected in single human sperm cells can reveal additional elemental bioavailability information in the identification of subpopulations of sperm cells, which was not revealed when only the mean mass of the element was analyzed as a whole population for a single human sperm sample. Dot plots show tracking of the entire defined sperm population of individual preparations of normal semen with or without a capacitation challenge. Arrows indicate subpopulations with clearly lower or higher Fe content of cells detected from the same sample population. Non-capacitated normal sperm (normal / original), capacitated normal sperm (normal / capacitated), non-capacitated oligoasthenospermia (abnormal / original), capacitated oligoasthenospermia (abnormal / capacitated). Data are expressed as mean ± SEM (n 3–30 individual samples). Two-way ANOVA, ****P<0.0001. [Figure 23B]We provide an association between elementomic mean mass frequency patterns and oligoasthenospermia risk. Using Fe elemental analysis as an example, we show that detailed frequency distribution analysis of the mean mass of Fe detected in single human sperm cells can reveal additional elemental bioavailability information in the identification of subpopulations of sperm cells, which was not revealed when only the mean mass of the elements was analyzed as a whole population for a single human sperm sample. Dot plots show the tracing of the entire defined sperm population of individual preparations of normal semen with or without a capacitation challenge. Arrows indicate subpopulations with clearly lower or higher Fe content of cells detected from the same sample population. Non-capacitated normal sperm (normal / original), capacitated normal sperm (normal / capacitated), non-capacitated oligoasthenospermia (abnormal / original), capacitated oligoasthenospermia (abnormal / capacitated). 2-way ANOVA, ****P<0.0001. [Figure 24A] Provides element-specific kinetic parameters of ICP-MS signals and their association with oligoasthenospermia risk.Provides an overview of sc-ICP-MS element signal kinetics of specific elements in original or stimulated normal or oligoasthenospermia human sperm capacitated by DGC, including pre-peak or post-peak residence times of ICP-MS signal spikes and pre-peak or post-peak tau constants for iron (Fe), copper (Cu) and zinc (Zn). [Figure 24B] We provide element-specific kinetic parameters of ICP-MS signals and their association with oligoasthenospermia risk. We provide an overview of sc-ICP-MS element signal kinetics (pre- or post-peak residence time and pre- or post-peak tau constants) of specific elements in human sperm with normal motility parameters, including Cr, Fe, Zn, Cu, Mn, and Se. Data are presented as mean ± SEM (n>3 individual samples). Two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Diagram 25]Examples of intrinsic elemental ICP-MS spike kinetics characteristics of essential elements, including Zn, Fe, Cu, Cr, and Mn, are provided based on sc-ICP-MS spike signals determined in different cell types, including single human sperm cells, cultured mouse epididymal epithelial DC2 cells, cultured human embryonic kidney HEK293T cells, cultured human cervical cancer HeLa cells, and cultured human gastric cancer SNU-1 cells. Parameters expressed in milliseconds include the dwell time of the signal spike of a particular element in a particular cell type, the dwell time consisting of pre-peak time (T0-to-Tpeak) and post-peak time (Tpeak-to-Tend). Data are presented as mean ± SEM (n>3 individual samples). Two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 26A-B] FIG. 26A shows the morphology of sperm after the spray chamber and sperm smear. To clarify the effect of the single cell ICP-MS loading system on sperm, the sperm morphology of sperm that passed through the nebulizer was compared. The figure shows that there was no significant difference in sperm morphology. FIG. 26B shows the morphology of sperm after the spray chamber and sperm smear. To clarify the effect of the single cell ICP-MS loading system on sperm, the sperm morphology of sperm that passed through the sperm smear was compared. The figure shows that there was no significant difference in sperm morphology. [Figure 27A] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Figure 2 shows Ca content (attograms per cell, 10-18) in single sperm cells at different time points of capacitation analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures show the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27B]Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Frequency distribution of calcium mass in single sperm cells at the indicated time points (0 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27C] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Frequency distribution of calcium mass in single sperm cells at the indicated time points (0.5 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27D] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Frequency distribution of calcium mass in single sperm cells at the indicated time points (1 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27E]Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Frequency distribution of calcium mass in single sperm cells at the indicated time points (1.5 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27F] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Frequency distribution of calcium mass in single sperm cells at the indicated time points (2 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27G] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm population of Pmca4 KO and WT mice. Data herein and in subsequent figures represent the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Fig. 27H] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm population of Pmca4 KO and WT mice. Data herein and in subsequent figures show the mean (±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 27I] Figure 1 provides dynamic calcium (Ca) flux analysis during capacitation showing calcium overload in live sterile sperm of Pmca4 KO and WT mice. Range of mean mass content of Ca (in attograms) in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. Data herein and in subsequent figures show the mean values ​​(±SD) of three mice per group. *P<0.05, **P<0.01, and ****P<0.0001, 2-way ANOVA. [Figure 28A] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Molar Zn in single sperm cells of Pmca4 KO and WT mice at different time points in capacitation conditions is provided and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28B] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Frequency distribution of Zn content of single sperm cells at the indicated time points (0 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28C] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Frequency distribution of Zn content of single sperm cells at the indicated time points (0.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28D]Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Frequency distribution of Zn content of single sperm cells at the indicated time points (1 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28E] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Frequency distribution of Zn content of single sperm cells at the indicated time points (1.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28F] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Frequency distribution of Zn content in single sperm cells at the indicated time points (2 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28G] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Fig. 28H] Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 28I]Dynamic zinc (Zn) flux analysis during capacitation of live mouse sperm as revealed by sc-ICP-MS. Range of mean mass content (in attograms) of zinc in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 from 2-way ANOVA. [Figure 29A] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Moles of Mn in single sperm cells of Pmca4 KO and WT mice at different time points of capacitation are provided and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29B] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Fe content in single sperm cells at the indicated time points (0 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29C] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Fe content in single sperm cells at the indicated time points (0.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29D] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Fe content in single sperm cells at the indicated time points (1 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29E]Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Fe content in single sperm cells at the indicated time points (1.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29F] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Fe content in single sperm cells at the indicated time points (2 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29G] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29H] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 29I] Dynamic iron (Fe) flux analysis during capacitation as revealed by sc-ICP-MS. Range of mean Fe mass content (in attograms) in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 30A]Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Moles of Mn in live single sperm cells of Pmca4 KO and WT mice at different time points of capacitation are provided and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30B] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Cu content in single sperm cells at the indicated time points (0 h) and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30C] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Cu content in single sperm cells at the indicated time points (0.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30D] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Cu content in single sperm cells at the indicated time points (1 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30E] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Cu content in single sperm cells at the indicated time points (1.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30F]Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Cu content in single sperm cells at the indicated time points (2 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30G] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30H] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 30I] Dynamic copper (Cu) flux analysis during capacitation as revealed by sc-ICP-MS. Range of average Cu mass content (in attograms) in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. ***P<0.001, 2-way ANOVA. [Figure 31A] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Figure 31A provides moles of Mn in live single sperm cells of Pmca4 KO and WT mice at different time points of capacitation and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 31B]Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Mn content in single sperm cells at the indicated time points (0 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 31C] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Mn content in single sperm cells at the indicated time points (0.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 31D] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Mn content in single sperm cells at the indicated time points (1 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 31E] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Mn content in single sperm cells at the indicated time points (1.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Fig. 31F] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of Mn content in single sperm cells at the indicated time points (2 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 31G]Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm population of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Fig. 31H] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Fig. 31I] Dynamic manganese (Mn) flux analysis during capacitation as revealed by sc-ICP-MS. Range of mean mass content (in attograms) of Mn in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05 and **P<0.01, 2-way ANOVA. [Figure 32A] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Molar Mn in live single sperm cells of Pmca4 KO and WT mice at different time points of capacitation is provided and analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Figure 32B] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of selenium (Se) content of single sperm cells at the indicated time points (0 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Figure 32C]Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of selenium (Se) content in single sperm cells at the indicated time points (0.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Fig. 32D] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of selenium (Se) content in single sperm cells at the indicated time points (1 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Figure 32E] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of selenium (Se) content in single sperm cells at the indicated time points (1.5 h) analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Fig. 32F] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Frequency distribution of selenium (Se) content in single sperm cells at the indicated time points (2 h) is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Fig. 32G] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Skewness of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Fig. 32H]Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Kurtosis of frequency distribution patterns is shown, analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Fig. 32I] Dynamic selenium (Se) flux analysis during capacitation as revealed by sc-ICP-MS. Range of average Se mass content (in attograms) in single live sperm cells at different time points under capacitation conditions analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice. *P<0.05, 2-way ANOVA. [Figure 33A] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of calcium is plotted against the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process. *P<0.05 by 2-way ANOVA. [Figure 33B] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of iron is plotted against the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process. *P<0.05 by 2-way ANOVA. [Figure 33C] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of copper is plotted against the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process. *P<0.05 by 2-way ANOVA. [Figure 33D] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of zinc is plotted against the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process. *P<0.05 by 2-way ANOVA. [Figure 33E] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of manganese to the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process is plotted. *P<0.05 by 2-way ANOVA. [Figure 33F] The ratio of the content of each element to the content of nickel (Ni) ions in the same single sperm population of Pmca4 KO and WT mice is shown. The molar content ratio of selenium is plotted against the molar content of Ni ions in single cells of different samples of KO and WT sperm during the capacitation process. *P<0.05 by 2-way ANOVA. [Figure 34A] Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Figure 34B] Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Figure 34C]Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Fig. 34D] Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Figure 34E] Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Fig. 34F]Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Figure 34G] Effect of cell density on the ICP-MS signal profile of single human sperm cells. Different cell densities from 3 x 10 per ml to 2 x 10 per ml of ICP-MS signal profiles of single human sperm cells during time-resolved scan times are shown (example shows signal for Fe element). ICP-MS intensities corresponding to ICP-MS ion counts of spike signals for determined elements. Insert shows distribution of ICP-MS spikes of Fe single events digitized at 50 μs sampling dwell time at various cell densities of human sperm. [Fig. 34H] FIG. 1 shows the effect of cell density on the ICP-MS signal profile of single human sperm cells. Graphical plot showing ICP-MS spike events per second independent of sperm density at densities below 2×106 per ml (arrows). [Fig. 34I] FIG. 1 shows the effect of cell density on the ICP-MS signal profile of single human sperm cells, and shows that spike events were also independent of the experimental time after the death of the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below at varying levels of detail in order to provide a substantial understanding of the present technology.

[0036] The section or subsection headings used herein are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described.

[0037] definition Definitions of certain terms used herein are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0038] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to a "cell" includes a combination of two or more cells, etc. Generally, the terminology used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are well known and commonly employed in the art.

[0039] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by a variation of + / - 15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%, as appropriate. It is understood, although not always expressly stated, that all numerical designations are preceded by the term "about". It is also understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0040] As used herein, the term "about" in reference to a number, unless otherwise stated or clear from the context, is generally interpreted as including numbers within 1%, 5%, or 10% in either direction (greater or less) of the number (except where such number is less than 0% or greater than 100% of its possible value).

[0041] As used herein, comparative terms used herein, such as high, low, increase, decrease, decrease, or any grammatical variation thereof, can refer to a particular variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1-fold, or about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50-fold, or about 60-fold, or about 70-fold, or about 80-fold, or about 90-fold, or about 100-fold, or more. In some embodiments, such a variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0042] "Optional" or "optionally" means that a situation described below may or may not occur, so that the description includes examples in which the situation occurs and examples in which the situation does not occur.

[0043] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0044] "Substantially" or "essentially" means nearly completely or completely, e.g., 95% or greater of some given amount. In some embodiments, "substantially" or "essentially" means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0045] In some embodiments, the use of "first," "second," "third," "fourth," or similar terms in a component name is used to distinguish and differentiate between multiple components that share a particular identity in their name. For example, a "first population of cells" and a "second population of cells" are used to distinguish between the two populations.

[0046] As used herein, the term "spermatozoon" or "sperm", or any grammatical variation of each thereof, refers to a male reproductive cell. In some embodiments, one or more sperm refer to a single male reproductive cell. In some embodiments, sperm refer to a plurality of male reproductive cells. Additionally or alternatively, the sperm may be live or dead. Additionally or alternatively, the sperm may be motile or immotile. Additionally or alternatively, the sperm may be capacitated or non-capacitated. Additionally or alternatively, the sperm may be present in a biological sample from a subject. In further embodiments, the sperm may be liquefied. In other embodiments, the sperm may be purified or concentrated, or both purified and concentrated. Additionally or alternatively, the sperm may be fixed. Additionally or alternatively, the sperm may be cryopreserved.

[0047] Sperm are produced in the testes and mature in the epididymis. Following this, they undergo a series of functional activities in the female duct before gaining the ability to fertilize an oocyte. Sperm capacitation is a functional activity essential for fertilization. Calcium ion influx is required for the capacitation process to occur (see, e.g., Navarrete et al., J Cell Physiol, 2015, 230, 1758-1769, and Ickowicz et al., Asian J Androl, 2012, 14, 816-821; Wassarman PM, Cell, 1999, 96:175-183).

[0048] ) Calcium efflux pump plasma membrane Ca in mice 2+Impairment of calcium homeostasis in sperm by genetic deletion of Pmca4-ATPase isoform 4 (Pmca4) leads to defective sperm motility and male infertility, accompanied by increased resting levels of intracellular calcium (see, e.g., Okunade et al., J Biol Chem, 2004, 279, 33742-33750, and Schuh et al., J Biol Chem, 2004, 279, 28220-28226).

[0049] "Metal element", "element" and "metal" are used interchangeably to refer to all elements in Groups 1-12 of the periodic table except hydrogen, and all elements in Groups 13-16 of the periodic table except all metalloid elements, C, N, P, O, S, and Se. In other words, when a metal element forms an inorganic compound with a halogen, the metal element becomes a cation. In some embodiments, the metal elements include metal elements in their elemental form, and metal elements in oxidized or reduced states, for example, when the metal element is combined with other elements in the form of a compound containing the metal element. For example, the metal element may be in the form of a hydrate, a salt, an oxide, and various polymorphs thereof.

[0050] The term "sperm capacitation", "capacitation", or any grammatical variation thereof, as used herein, refers to the process of sperm undergoing acrosomal exocytosis, allowing them to acquire the ability to fertilize an egg, such as by binding and penetrating through the zona pellucida of an unfertilized egg (see, e.g., Wassarman PM, Cell, 1999, 96:175-183). Completion of capacitation is manifested by the ability of sperm to bind to the zona pellucida and undergo a ligand-induced acrosome reaction. In some embodiments, capacitation refers to an in vivo process. For example, sperm capacitation occurs naturally throughout the female reproductive tract. In some embodiments, capacitation refers to an ex vivo or in vitro process. For example, assisted reproductive technologies perform in vitro capacitation techniques to improve the chances of successful fertilization. Different techniques exist for performing the capacitation step: simple wash, transfer (swim-up), density gradient, and filtration.

[0051] In some embodiments, a method for performing in vitro capacitation comprises, consists essentially of, or even consists of one or more of the following: (1) Simple wash: refers to washing to remove seminal plasma from a sample containing sperm. In some embodiments, the sample is centrifuged and then the supernatant is removed. (2) Migration (swim up): refers to the selection of sperm cells based on their ability to migrate ("swim") upward from the bottom to the top of the tube. Non-limiting examples are provided herein. First, centrifugation is performed to remove the seminal plasma. Then, 0.5-1 ml of culture medium or another buffer is added to the top, and after an incubation period at 37°C, the best motile sperm are allowed to rise from the bottom to the top of the tube (moving healthy sperm into the culture medium), and the upper layer is collected to obtain a sperm-rich fraction. (3) Density gradient: refers to density gradient centrifugation of a sample containing sperm cells. Non-limiting examples are provided herein. A tube is filled with layers of liquids of different densities, semen is placed in the top layer, and then the tube goes through centrifugation to filter out cell debris and non-motile cells. After centrifugation, healthy sperm are in the bottom layer of the liquid in the tube, while debris and non-motile sperm are in the upper layer, and finally, all cells reach the bottom, but more motile cells reach it earlier. This procedure is often simply called the "Percoll method" because Percoll was frequently used as the density medium, but other density media can also be used. (4) Filtration: refers to filtering a sample containing sperm cells. A non-limiting example includes, consists essentially of, or even consists of a filter that does not allow all sperm to pass through; only sperm with better motility will pass through the filter. (5) Other: in vitro fertilization (IVF), physiological intracytoplasmic sperm injection (PICSI), sperm penetration assay (SPA), magnetic activated cell sorting (MACS) or microfluidic chips, each of which optionally obtains motile sperm.

[0052] Methods for determining sperm capacitation are known in the art, for example, the most common sperm-zona binding tests currently in use are the hemizona assay (or HZA) and the competitive intact-zona binding assay. The hemizona assay measures the ability of sperm to undergo capacitation and bind to oocytes. Sperm are incubated with dead oocytes surrounded by a zona pellucida and a cellular coating of the oocyte. Capacitated sperm bind to the zona and the number of sperm bindings is counted by microscope. This number correlates with the number of normally capacitated sperm in the sample and the fertilization rate of the sperm sample. See, for example, Cross et al., Gamete Res. 1986; 15: 213-26, and Wassarman PM, Cell, 1999, 96: 175-183).

[0053] Human tubal fluid (HTF) buffer refers to a synthetic solution that mimics the composition of the fluid found in the human fallopian tube. It is suitable for procedures such as the retrieval, handling and transfer of human gametes and embryos. In some embodiments, HTF buffer uses a sodium bicarbonate buffer system, which is suitable for those procedures that require the use of a carbon dioxide atmosphere during incubation. In some embodiments, HTF buffer as used herein also refers to modified HTF buffers that use a different buffer system, such as a combined sodium bicarbonate / HEPES buffer. In some embodiments, the buffer system provides for the maintenance of a physiological pH (e.g., 7.2-7.4).

[0054] As used herein, the term liquefaction or any grammatical variations thereof, such as liquefied, refers to a process in which the gel formed by proteins from the seminal vesicles is broken down and the semen becomes more liquid. In some embodiments, placing the semen sample at room temperature or 37° C. for about 15 minutes to about 60 minutes (such as about 30 minutes to about 60 minutes) liquefies the sample. In further embodiments, continuous gentle mixing or rotation of the sample container on a two-dimensional shaker during liquefaction can help to generate a homogenous sample. Additionally or alternatively, mechanical mixing (such as repeated pipetting or gentle passage through a blunt gauge 18 or gauge 19 needle attached to a syringe) or enzymatic digestion (such as digestion with bromelain or other proteolytic enzymes) or both can be used for liquefaction.

[0055] "Fixation" refers to a process that maintains the structure of cells and / or subcellular components, such as organelles (e.g., nuclei). Fixation modifies cellular components in chemical or biological structure, for example, by cross-linking them. Fixation allows whole cells and organelles to resist lysis. "Fixative" refers to an agent, such as a chemical or biological reagent, that fixes cells. Fixatives can disable cellular proteolytic enzymes and nucleases. Examples of fixatives include aldehydes (e.g., formaldehyde, or paraformaldehyde (PFA)), alcohols, and oxidizing agents. Examples of suitable fixatives are provided in U.S. Patent Application Publication No. 2010 / 0184069, filed January 19, 2010, and U.S. Patent Application Publication No. 2010 / 209930, filed February 11, 2010.

[0056] As used herein, the term "cryopreservation" refers to the stable maintenance of cells for extended periods of time via freezing. In some embodiments, cell cryopreservation is for freezing and preserving cells before they lose their inherent properties, or for using them as needed, or for both. Cell cryopreservation can be performed by contacting the cells to be cryopreserved with a cryoprotectant, and optionally a cell culture medium, to prevent cell damage caused by the cryoprotectant, thereby improving the safety and stability of the cryopreservation of cells. As used herein, the term "cryoprotectant" refers to a substance used when cells are stored at temperatures below 4°C, or at ultra-low temperatures between -80°C and -200°C. In particular, this substance can minimize the formation of ice crystals and cell damage due to ionic and osmotic imbalances that inevitably accompany the freezing and thawing process.

[0057] As used herein with respect to molecules, the terms "isolated," "isolating," or "isolation," and "purified," "purifying," or "purification" do not refer to absolute purity. Rather, "purified," "purifying," or "purification" refers to a substance in a composition that contains fewer species of the same class (e.g., cell types) other than the substance of interest, as compared to the sample from which it is derived.

[0058] As used herein, CASA is an acronym for computer-assisted sperm analysis or computer-aided sperm analysis, and refers to the process of analyzing the concentration, motility, cell morphology, or any combination thereof, of a semen sample with the aid of a computer. Several manufacturers, such as MICROPTIC SL and Hamilton Thorne, produce CASA systems. Furthermore, the use of CASA to measure sperm motility and concentration is described in sections 3.5.2 and 3.5.3 of WHO, 2010, respectively.

[0059] As used herein, the term "IVF" or "in vitro fertilization" refers to the fertilization of an oocyte with sperm outside of an organism. IVF may also refer to the technique of mixing an oocyte and sperm in a laboratory to achieve fertilization.

[0060] As used herein, the term "intracytoplasmic sperm injection" (ICSI) refers to a process in which sperm cells are directly injected into an egg, such as a human egg, to promote fertilization of the egg and zygote formation. Sperm cells can be injected into an egg, for example, by penetrating the egg cell membrane with a microinjector to deliver the sperm cells directly into the egg's cytoplasm. ICSI procedures useful in conjunction with the compositions and methods described herein are known in the art and are described, for example, in WO2013 / 158658, WO2008 / 051620, and WO2000 / 009674, among others.

[0061] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) refers to a type of mass spectrometry (MS) that uses an inductively coupled plasma (ICP) to ionize a sample. It is known and used for its ability to detect metals and some nonmetals in liquid samples at very low concentrations. When a sample (such as a single cell as disclosed herein) enters the ICP, it is vaporized, atomized, and ionized to form a cloud of elemental ions. The ions generated are directed from the ICP to the mass analyzer through a pressure reduction interface that regulates the pressure difference between the atmospheric pressure ICP and the low pressure (e.g., 10-6 mbar) mass analyzer. In some embodiments, ion optics are used to efficiently transmit the ions to the mass analyzer. The mass analyzer separates the ions according to their mass-to-charge ratio (m / Q) using electric or magnetic fields, or both, before hitting the detector. The data generated indicates the number of ions recorded for each m / Q. The m / Q can be used to determine the elemental identity of the ions, and the number of ions to determine elemental concentration. The cloud of elemental ions generated from a sample in an ICP source produces a very fast transient signal (referred to herein as a "signal spike" or "spike") that has a total duration (referred to herein as a "dwell time"). While scanning analyzers typically target one or two elements, time-of-flight (TOF) mass analyzers can record the entire mass spectrum (all m / Q values) for each sample. For any recorded isotope (m / Q value), the total ion signal observed during the duration of the transient particle signal is proportional to the mass of that element in the sample. The frequency of particle events (transient signal spikes) detected by ICP-MS is proportional to the particle number concentration in the introduced liquid sample. The continuous signal region that does not contain spikes (single particle detection events) represents the concentration of the sample fraction present in dissolved form. The terms "peak" and "spectral peak" refer to peaks in the output from a mass spectrum, such as the peaks of a spike.

[0062] In some embodiments, the dynamic parameters refer to parameters that change over time of interest, rather than absolute parameters at a particular time of interest, such as the rising tau constant before the peak (i.e., the amount of time that elapses from the start of the spike to the peak in a manner according to an alpha exponential algorithm, also called a standard exponential function; the fitting of the function algorithm should not be limited to this function only), the dynamic area before the peak (i.e., the area under the spike before the peak), the tailing tau constant after the peak (i.e., the amount of time that elapses from the peak to the end of the spike in a manner according to an alpha exponential algorithm, also called a standard exponential function; however, the fitting of the function algorithm should not be limited to this function only), the dynamic area after the peak (i.e., the area under the spike after the peak), and the area of ​​the peak or spike (i.e., the area under the spike). See the illustration in FIG. 5.

[0063] In some embodiments, the alpha exponential function comprises the following algorithm:

number

[0064] In some embodiments, kinetic parameters refer to parameters describing the dynamics of a spike, such as dwell time (i.e., the amount of time elapsed during a spike), dwell time before peak (i.e., the amount of time elapsed during the spike and before the peak), dwell time after peak (i.e., the amount of time elapsed during the spike and after the peak), and peak time (i.e., the amount of time elapsed for a spike to reach a peak proportional to the spike dwell time). See illustration in FIG. 5. In some embodiments, the term "kinetic parameters" is used interchangeably with "dynamic parameters."

[0065] As used herein, a first level that is "comparable" to a second level refers to the first level being substantially similar compared to the second level. For example, the first level is about 50% to about 2 times the second level (or any percentage, factor, or range therebetween). In some embodiments, the first level is about 80% to about 120% of the second level (or any percentage, factor, or range therebetween, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, or about 110%). In some embodiments, a first level that corresponds to a second level refers to a first level that is comparable to the second level. Additionally or alternatively, a first range that corresponds to a second range refers to an upper or lower limit, or both an upper and lower limit, of a first range that is comparable to the second range.

[0066] As used herein, a predetermined level, a predetermined range, a reference level, a reference range, and a reference refer to a range of levels in a control sample. In some cases, the control sample is obtained from a healthy subject, e.g., a subject that does not have one or more of diseases or conditions. In a further example, the control sample is a reference sample specific to a laboratory facility, and the predetermined level is established for a particular assay of interest by the laboratory facility that performs the particular assay of interest. In some cases, the predetermined level is measured using the samples disclosed herein. Those skilled in the art will understand that the level is influenced by the assay, the age of the subject, and the health of the subject.

[0067] As used herein, a biological sample, or sample, is obtained from a subject. Exemplary samples include cell samples, cell-derived particles (e.g., sperm-derived exosomes, non-sperm-derived exosomes, epididymosomes, membrane-enveloped particles, lipid-enveloped biological particles, cytoplasmic droplets, and / or organelles such as mitochondria, lysosomes, endosomes, endoplasmic reticulum, smooth endoplasmic reticulum, rough endoplasmic reticulum, fimbria, primary cilia, ribosomes, Golgi apparatus, nuclei, chromatin, chromosomes, mesosomes, peroxisomes, microtubules, actin filaments, and / or intermediate filaments), tissue samples, and the like. Examples of liquid samples that may be used include, but are not limited to, liquid samples such as tumor biopsies, blood, and other liquid samples of biological origin, including, but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / flushings, synovial fluid, mucosal secretions, stool water, pancreatic juice, washings from sinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, or umbilical cord blood. In some cases, the sample is a semen sample. In some cases, the sample is a cell-derived particle (e.g., a sperm-derived exosome, a non-sperm-derived exosome, an epididymosome, a membrane-enveloped particle, a lipid-enveloped biological particle, a cytoplasmic droplet, and / or a cellular organelle such as a mitochondria, a lysosome, an endosome, an endoplasmic reticulum, a smooth endoplasmic reticulum, a rough endoplasmic reticulum, a cilium, a primary cilium, a ribosome, a Golgi apparatus, a nucleus, a chromatin, a chromosome, a mesosome, a peroxisome, a microtubule, an actin filament, and / or an intermediate filament).

[0068] As used herein, the term "animal" refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term "mammal" includes both human and non-human mammals.

[0069] The terms "subject," "host," "individual," and "patient," as used interchangeably herein, refer to an animal, typically a mammal. Any suitable mammal may be treated by the methods described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., adult, teen, child, infant, or mammal in utero). The mammal may be male or female. In some embodiments, the subject is a human. In some embodiments, the subject has, is diagnosed as having, or is suspected of having a disease.

[0070] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing a symptom or disease from occurring in a subject who is predisposed to the disease or who does not yet exhibit symptoms thereof, (2) inhibiting or arresting the onset of the disease, or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the severity of a condition (including a disease), a stable (i.e., non-worsening) stage of a condition (including a disease), delay or slowing of a condition (including a disease), progression, amelioration, or alleviation, stage, and remission (partial or complete) of a condition (including a disease). In one aspect, treatment excludes prevention.

[0071] In some embodiments, the term "disease" or "disorder" as used herein refers to infertility, a condition diagnosed as infertility, a condition suspected of having infertility, or a condition at high risk of having infertility. In some embodiments, the disease is in males.

[0072] As used herein, the term "infertility" refers to the lack or reduced ability to conceive or bear offspring. Infertility can be present in either males or females. As used herein, male infertility refers to a male's inability to induce pregnancy in a fertile female. Male infertility is generally due to a lack of semen, and semen quality may be used as a surrogate measure of male fertility. In some embodiments, infertility comprises, consists essentially of, or even consists of idiopathic infertility, such as asthenozoospermia, asthenozoospermia, necrospermia, oligozoospermia, oligoasthenozoospermia, oligoasthenozoospermia, or teratozoospermia.

[0073] As used herein, the term "fertile" refers to a male subject who has motile or viable sperm, or both motile and viable sperm, and therefore has a significant potential to initiate pregnancy. In some embodiments, the term "infertility" refers to the inability to establish a clinical pregnancy after 12 months of regular, unprotected intercourse.

[0074] The term "unexplained" or "idiopathic" infertility applies to one or both subjects of all couples who have not conceived within at least one year, who have no clinical explanation, and whose fertility testing of both partners has not revealed an identifiable cause. A human couple or any partner of a couple is inadequately diagnosed as having "unexplained" or "idiopathic" infertility if standard fertility evaluations have found the couple to be "normal" (e.g., female - ovulation and normal postcoital examination, regular endometrial biopsy, hysterosalpingography, laparoscopy; male - "normal" sperm analysis, at least two sperm concentrations greater than 20 million / ml, total sperm count greater than 40 million, sperm motility greater than 50%, and normal morphology of more than 30% of the sperm) and if the couple has a history of involuntary infertility for at least one year. Such couples often undergo many invasive, protracted and expensive attempts at assisted reproductive technology in pursuit of conception, such as IVF.

[0075] Asthenozoospermia refers to a condition characterized by reduced sperm motility compared to the average level or range of a healthy subject or a population of healthy subjects. In some embodiments, male subjects with asthenozoospermia exhibit a percentage of progressively motile (PR) sperm below a predetermined level.

[0076] Asthenozoospermia refers to a condition characterized by reduced sperm motility and abnormal sperm morphology compared to the average level or range of healthy subjects or a population of healthy subjects. In some embodiments, male subjects with asthenozoospermia exhibit a percentage of both progressive motility (PR) and morphologically normal sperm below a predetermined level.

[0077] Azoospermia refers to a condition in which sperm in semen are immobile or dead, or both. In some embodiments, male subjects with azoospermia exhibit lower viability, higher immobility, or both lower viability and higher immobility of sperm in ejaculate compared to healthy subjects, or the average level or range of a population of healthy subjects.

[0078] Oligoteratozoospermia refers to a combination of oligozoospermia and teratozoospermia. In some embodiments, male subjects with oligozoospermia exhibit a total sperm count (or concentration) below a certain level, and a percentage of morphologically normal sperm.

[0079] Teratozoospermia refers to a condition characterized by the presence of morphologically abnormal sperm. In some embodiments, male subjects with teratozoospermia exhibit a percentage of morphologically normal sperm below a predetermined level.

[0080] Oligozoospermia refers to a condition in which a lower number of sperm are produced in an ejaculate compared to the average level or range of a healthy subject or a population of healthy subjects. In some embodiments, a male subject with oligozoospermia exhibits a total sperm count (or concentration) below a predetermined level.

[0081] Oligoasthenozoospermia refers to a combination of oligospermia and asthenozoospermia. In some embodiments, male subjects with oligoasthenozoospermia exhibit a total sperm count (or concentration) and a percentage of progressively motile (PR) sperm below a certain level.

[0082] Oligoasthenozoospermia refers to a combination of oligospermia, asthenozoospermia and teratozoospermia. In some embodiments, male subjects with oligoasthenozoospermia exhibit a total sperm count (or concentration) below a certain level, as well as a percentage of both progressively motile (PR) and morphologically normal sperm.

[0083] A simple system for grading motility is recommended by WHO, 2010, which distinguishes sperm with progressive or non-progressive motility from immotile sperm. The motility of each sperm is graded as follows: Progressive motility (PR): Sperm are actively moving either in a straight line or in a large circle, regardless of speed. Non-progressive motility (NP): All other patterns of motility without progress, e.g., swimming in small circles, where flagellar forces barely displace the head, or where only flagellar beats can be observed. Immotile (IM): No movement. In some embodiments, the predetermined level for PR is about 31% to about 34%, or any percentage or range therebetween, e.g., about 31%, or about 32%, or about 33%, or about 34%. In some embodiments, the predetermined level for total motility (PR and NP) is about 38% to about 42%, or any percentage or range therebetween, e.g., about 38%, or about 39%, or about 40%, or about 41%, or about 42%.

[0084] In some embodiments, the predetermined level for vitality (such as membrane-intact sperm) is about 55% to about 63%, or any percentage or range therebetween, for example, about 55%, or about 56%, or about 57%, or about 58%, or about 59%, or about 60%, or about 61%, or about 62%, or about 63%.

[0085] In some embodiments, the predetermined level for sperm concentration is about 12×10 per ml 6 ~16×10 6 spermatozoa, or any concentration or range therebetween, e.g., about 12×10 sperm per ml 6 spermatozoa or approximately 13 x 10 per ml 6 spermatozoa or approximately 14 x 10 per ml 6 spermatozoa or approximately 15 x 10 per ml 6 spermatozoa or approximately 16 x 10 per ml 6 Each sperm cell is divided into 10 sperm.

[0086] In some embodiments, the predetermined level for total sperm count is about 33×10 per ejaculation.6 ~46×10 6 spermatozoa per ejaculate, or any concentration or range therebetween, e.g., about 33×10 6 spermatozoa or approximately 34 x 10 per ejaculate 6 spermatozoa or approximately 35 x 10 per ejaculate 6 spermatozoa or approximately 36 x 10 per ejaculate 6 spermatozoa or approximately 37 x 10 per ejaculate 6 spermatozoa or approximately 38 x 10 per ejaculate 6 spermatozoa or approximately 39 x 10 per ejaculate 6 spermatozoa, or approximately 40 x 10 per ejaculate 6 spermatozoa, or approximately 41 x 10 per ejaculate 6 spermatozoa, or approximately 42 x 10 per ejaculate 6 spermatozoa or approximately 43 x 10 per ejaculate 6 spermatozoa or approximately 44 x 10 per ejaculate 6 spermatozoa or approximately 45 x 10 per ejaculate 6 spermatozoa or approximately 46 x 10 per ejaculate 6 Each sperm cell is divided into 10 sperm.

[0087] In some embodiments, the predetermined level for morphologically normal sperm in an ejaculate is about 3% to about 4%, or any percentage or range therebetween, e.g., about 3%, or about 4%.

[0088] Infertility therapy refers to the treatment of infertility, for example, a method, composition, active ingredient, or any combination thereof. Various infertility therapies include, but are not limited to, assisted reproductive technologies (ART), in vitro fertilization (IVF), ovarian hyperstimulation, controlled ovarian hyperstimulation, natural in vitro fertilization cycles, terminal maturation induction, transvaginal oocyte retrieval, egg and sperm preparation, co-inoculation, embryo culture, adjuvants, cycle stimulation therapy, follicle stimulating hormone (FSH) therapy, microdose gonadotropin releasing hormone antagonist (GnRHa) flare therapy, antagonist (e.g., GnRHant) therapy, intracytoplasmic sperm injection (ICSI), enhanced intracytoplasmic sperm injection, mitochondrial enhanced intracytoplasmic sperm injection, and related female germline stem cell therapies (e.g., AUGMENT (service mark), OVAPRIME (service mark), and OVATURE (service mark) therapies offered by OvaScience, Inc. of Waltham, Mass.), adoption, and the like.

[0089] MODES FOR CARRYING OUT THE DISCLOSURE Elementomics in Biomedical Sciences: Metals play essential functions in life. One third of proteins are considered to be metalloproteins, which require a metal cofactor, which is usually a transition metal, e.g., copper (Cu), iron (Fe), cobalt (Co), chromium (Cr) or manganese (Mn) (Nolan, 2016, Science 352, 1055-1056, and Waldron et al., 2009, Nature 460, 823-830), as well as the element selenium (Se) (Green, 2018, Cell 172, 389-390, and Kieliszek et al., 2021, Biol Trace Elem Res). Most of these transition metals or elements are redox active in biological systems or bind to macromolecules that can be controlled by redox potential. Metal cofactors are also required for proper folding and other biological functions of proteins, with zinc (Zn) and calcium (Ca) being among the most common cofactors (Bushmarina et al., 2006, Protein Sci 15, 659-671). Therefore, understanding the totality of metals and metalloids and elemental species within a cellular compartment or a cell or tissue type, i.e., elemental profiling, is important and has received increasing attention in the field of life sciences (Miyashita et al., 2017, In Metallomics: Recent Analytical Techniques and Applications, Y. Ogra, and T. Hirata, eds. (Tokyo: Springer Japan), pp. 107-124; Mounicou et al., 2009, Chem Soc Rev 38, 1119-1138; Nelson, 1999, Embo J 18, 4361-4371; Nolan and Waldron et al., 2009, Nature 460, 823-830). Characterization of the elemental profile of a given biological system requires a comprehensive analysis in a systematic approach by studying the entire content, speciation, and localization of elements, as well as the associated biomolecular profile of how the elements are sensed, stored, or used.For example, the bioavailability of a series of transition metals, such as Cu, Fe, and Zn, and the intracellular concentrations of their associated metallotheoneins, are believed to correlate with several protein-level expressions and their distribution among various cellular compartments (Calvo et al., 2017, IUBMB Life 69, 236-245; Outten and O'Halloran, 2001, Science 292, 2488-2492; Ruttkay-Nedecky et al., 2013, Int J Mol Sci 14, 6044-6066; Sakulsak, 2012, International Journal of Morphology 30, 1007-1012; and Waldron et al., 2009, Nature 460, 823-830). On the other hand, albumin and transferrin are essential metal transporters in extracellular fluid or plasma, and are internalized and transported to the desired cellular compartment through the process of endocytosis as needed (Kawabata, 2019, Free Radic Biol Med 133, 46-54, and Waldron et al., 2009, Nature 460, 823-830). Thus, the distribution of these metals and their metalloproteins can be tightly controlled and functionally interconnected (Chang, 2015, Nat Chem Biol 11, 744-747; Duncan, 2009, Metal Ions in Life Sciences Vol. 5. Edited by Astrid Sigel, Helmut Sigel and Roland KOSigel. 48, 7966-7967; Krezel and Maret, 2017, Int J Mol Sci 18; Mounicou et al.; Nelson; Sakulsak; and Tvrda et al., 2015, J Assist Reprod Genet, 2015, 32, 3-16). Each biological system is thought to be engineered into a specific set of element-protein partnerships.With increasing evidence of the biomineral functions of metals in reproductive biology, the importance of metal ion homeostasis in sperm function and male fertility is well documented. However, the mechanisms by which each metal-protein partnership occurs in a given biological system such as sperm and how this partnership is maintained under physiological conditions and dysregulated under pathophysiological conditions remain largely unknown.

[0090] Recent advances in single-cell elemental chemistry: Intact sperm function is the basis of male reproductive health. Biometals play an essential role in sperm function and male fertility, and imbalance in trace element homeostasis is one of the key factors leading to male infertility (Ali et al., 2017, Biol Trace Elem Res 175, 244-253; Mirnamniha et al., 2019, Rev Environ Health 34, 339-348; Schmid et al., Human Reproduction, 2013, 28, 274-282; and Tvrda et al.). For example, dynamic calcium concentration in the state of sperm capacitation (Navarrete et al., J Cell Physiol, 2015, 230, 1758-1769). Moreover, the effects of different elements such as Zn and Se can improve male fertility (Colagar et al., 2009, Nutr Res 29, 82-88, and Hawkes and Turek, 2001, J Androl 22, 764-772), while Cd can weaken male fertility, and it has also been reported that different concentrations of Nickel (Ni) have a dual effect on sperm motility for post-capacitation sperm (de Angelis et al., 2017, Reprod Toxicol 73, 105-127, Kumar and Sharma, 2019, Rev Environ Health 34, 327-338, and Mirnamniha et al.). These inorganic elements exert their functions in biological systems due to their unique physiochemical properties and quantities. Macroscopic amounts of metals are usually metals that are stable in an ionic state, such as potassium (K), sodium (Na), Ca, and magnesium (Mg) (Mirnamniha et al.).Those elements that play important roles at moderate levels in reproduction and sperm biology are usually transition metals such as Fe, Cu, and Zn, while some other essential elements such as Co, Cr, and Mn, as well as rare earth elements, can induce various physiological effects at very small amounts (Marzec-Wroblewska et al., Arch Environ Contam Toxicol, 2015, 69, 191-201; Mirnamniha et al., and Tvrda et al.). Several metalloid or heavy metal contaminations, such as arsenic (As), cadmium (Cd), mercury (Hg) and lead (Pd), are known to have toxic effects on sperm function (Inhorn et al., 2008, Reprod Toxicol 25, 203-212; Wang et al., 2016, Sci Total Environ 571, 307-313; and Wang et al., 2017, Environ Pollut 224, 224-234).

[0091] In fact, ion content can also be a criterion for evaluating sperm quality. Many studies have reported that cadmium and nickel weaken male fertility, selenium can improve fertility, and calcium is an important essential ion for sperm capacitation. See, for example, Ingold et al., Kasperczyk et al., Li et al., 2012a, Li et al., 2012b, Marzec-Wroblewska et al., Schmid et al., Tvrda et al., and Zhao et al., 2017. Therefore, detecting the ion spectrum of a single sperm provides a new angle and direction for sperm quality evaluation methods. On the other hand, clinical ion spectrum analysis methods for single cells are still lacking. Therefore, the establishment of a method for detecting the ion spectrum of a single sperm and a single cell is very important to clinically improve sperm quality, cell function, and provide reference values.

[0092] Interestingly, there is growing evidence that sperm dysfunction is associated with elemental imbalance (see, for example, Schmid et al., Marzec-Wroblewska et al., and Kasperczyk et al., J Trace Elem Med Biol, 2015, 30, 153-159). Studies have reported that disordered trace metals such as cadmium, manganese and nickel are associated with cases of weakened fertility in men (see, for example, Li et al., 2012a, Li et al., 2012b, BMC Public Health, 2012, 12, 919, and Zhao et al., PLoS One, 2017, 12, e0186727), while selenium can improve fertility (see, for example, Ingold et al., Cell, 2018, 172, 409-422 e421). Moreover, an imbalance between iron and copper homeostasis can cause increased oxidative stress, thus affecting sperm cell viability and even sperm cell death (see, e.g., Tvrda et al., J Assist Reprod Genet, 2015, 32, 3-16). Therefore, elemental ion content can be selected as a standard parameter for evaluating sperm quality. However, a method for monitoring dynamic elemental changes during sperm capacitation at the single cell level has not yet been reported.

[0093] Although it is ambitious, a comprehensive network of element-protein partnerships and their spatiotemporal distribution in sperm cells and reproductive tissue compartments, as well as how this network is maintained and how element-protein partnerships interact cooperatively, would allow a comprehensive understanding of elemental biology in sperm and reproduction. However, no methods have been reported for detecting metal ion concentrations in single sperm cells that evaluate functional activity, the so-called metallomics. The bioavailability of elements, or in other terms, elementomics, especially at the single cell level, is considered to be the first step to understand a specific set of element-protein partnerships in sperm cells. Thus, in some embodiments, the objective of the present disclosure was to develop and validate a method for simultaneously determining the content of multiple elements and the analysis of elemental characteristics in human sperm, i.e., elementomics profiling.

[0094] Need for a method of elemental analysis of sperm at single cell level: The impetus for the development of the technology disclosed herein stems from the major shortcomings of existing elemental determination methods for biological materials, which require large sample volumes or have low detection sensitivity, few detected elements, long detection times, and harsh sample preparation procedures that are very different from the physiological conditions for inorganic elements functioning in biological systems. For example, these analytical methods are usually applied to samples in solution and involve digesting organic samples into simpler inorganic forms in solution using strong acids, which are very different from the physiological conditions for inorganic elements functioning in biological systems. Furthermore, the major shortcomings of existing elemental determination methods, the "flame method" technology, are low sensitivity, few detected elements, and long detection times. Available methods for analyzing elements in biological materials include potentiometric, voltametric, atomic spectroscopy, and X-ray fluorescence techniques (Brown and Milton, 2005, Trac-Trend Anal Chem 24, 266-274, and Zhao et al., 2014, Trends in plant science 19, 183-192). In clinical laboratories, atomic spectroscopy is a common technique used for elemental analysis of human biological materials, including flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), and inductively coupled plasma atomic emission spectrometry (ICP-MS) (Bulska and Ruszczynska, 2017, Physical Sciences Reviews 2).

[0095] ICP-MS techniques have been applied to assess the elemental composition of digested samples, including digested human sperm and semen (Ali et al.; Li et al., 2012a, Biol Trace Elem Res, 2012, 148, 1-6; Marzec-Wroblewska et al.; Sorensen et al., 1999, Molecular human reproduction 5, 331-337; Wang et al., 2016, and Wang et al., 2017). Although conventional ICP-MS techniques have been applied to sperm elemental composition assessment (see, for example, Sorensen et al., 1999), elemental analysis at the single-cell level cannot be achieved without recent technological advances (Cao et al., 2019, Talanta 206, 120-174; Ho and Chan, 2010, J Anal Atom Spectrom 25, 1114-1122; Laborda et al., 2014, Anal Chem 86, 2270-2278; Liu et al., 2010, Talanta 83, 48-54; Meyer et al., 2018, Metallomics 10, 73-76; Miyashita et al.; Mueller et al., 2014, Anal Bioanal Chem 406, 6963-6977; and Wang et al., 2015, Analyst 140, 523-531). The latest development of single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) technology allows direct detection of elemental content in single cells. Several studies have applied sc-ICP-MS for rapid and simultaneous assessment of the composition of multiple elements at the single particle or single mammalian cell level (Bandura et al., 2009, Anal Chem 81, 6813-6822, Cao et al., Ho and Chan, Meyer et al., Miyashita et al., Mueller et al., and Wang et al., 2015).The sc-ICP-MS technique uses a single-cell nebulizer to atomize the cell suspension into small droplets containing single cells, which are then placed into a single-cell fog chamber, and the single-cell level analysis is realized through a fast analysis computer software module. However, the majority of cells verified are all somatic cells, including red blood cells and human cell lines, and for highly morphologically polarized cells such as sperm or cells isolated from body fluids, no relevant detection methods exist. So far, it has not been applied to the analysis of single human sperm.

[0096] As illustrated in the Examples, the sc-ICP-MS technique was utilized to verify the feasibility of elemental characterization for the evaluation of normal human sperm and oligoasthenospermia at the single cell level. Cultured somatic cells commonly used in laboratories, such as cancer cells, were also included in the analysis for comparison purposes. For this purpose, 18 elements were measured using sc-ICP-MS in single cells of a given biological sample for validation. It was found that different cell types not only have different single-cell elemental characterization profiles, but also have unique dynamic kinetics of sc-ICP-MS signal spikes of specific elements. In highly polarized sperm, no differences were observed in the average mass content of elements in single normal sperm and oligoasthenospermia, except for Ca. However, further analysis on the frequency distribution of the average mass content and inter-element correlations revealed significant differences of certain elements measured in normal and abnormal sperm, such as the inverse correlation between the essential elements K and Na, and the toxic elements Cd or Pb, which were inversely correlated with the essential element Ca. Furthermore, by using more sophisticated analysis, the unique dynamic kinetics of single sc-ICP-MS spike signals of specific elements (such as Fe and Cu) can be determined in human sperm. These elementomic signatures of Fe and Cu in normal human sperm were significantly different from those in oligoasthenospermia under normal and capacitation conditions. The elementomic signatures of somatic cells, including cancer cells, were also found to be different from those of human sperm. It was then proposed that this previously unreported single-cell element-specific kinetic signature in a specific cell type, such as human sperm, can be used as an elementomic signature to evaluate the physiological and pathophysiological status of a specific cell type in a given biological sample from a subject. In summary, the disclosure herein explores the potential applications of single-cell ICP-MS elementomic signature analysis for sperm functional quality, male fertility, and even the diagnosis and prognosis of medical diseases and conditions such as cancer.

[0097] In some embodiments, as a proof-of-concept demonstration, we utilize single-cell inductively coupled plasma mass spectrometry (ICP-MS) technology to measure the content of inorganic metal ions in live single sperm cells undergoing the capacitation process. Using a male sterile calcium efflux pump Pmca4 KO mouse model, we demonstrate, for the first time, the use of single-cell ICP-MS technology to evaluate the elemental composition of single sperm cells during the in vitro capacitation process to assess sperm functional activity and correlate sperm quality of individual animals. Thus, the disclosure herein provides a new method and metallomics-based diagnostic tool for the assessment of sperm quality and male fertility. Using sterile Pmca4 KO sperm cells, we monitored dynamic intracellular calcium levels as well as levels of other elements, such as, but not limited to, Zn, Fe, Cu, Mn, and Se (Figures 27A-27I-Figures 33A-33F). This previously unreported method will be useful for experimental and clinical assessment of metal composition during dynamic biological processes and to identify normal and abnormal sperm metallomic properties at the single-cell level.

[0098] The scientific findings disclosed herein provide dynamic and kinetic characterization of element-specific sc-ICP-MS signals of specific cells, thereby solving the technical problem of assessing cellular functions using multimodal analysis of single-cell ion spectroscopy, for example, in a clinical setting. This technique can be broadly used for quality testing of specific cells, including but not limited to the following cells and their associated functions: (1) To provide reference parameters for sperm function, infertility, and offspring health; (2) To provide reference parameters for egg cell function and infertility; (3) to provide reference parameters for granulocyte function and infertility; (4) To provide reference parameters for blood leukocyte function, physiological health and pathological diagnosis; (5) Providing reference parameters for the diagnosis of physiological health and pathological conditions of cells isolated from urine; (6) Providing reference parameters for the diagnosis of physiological health and pathological conditions of cells (including somatic and prokaryotic cells) isolated from stool; or (7) To provide reference parameters for the diagnosis of physiological health and pathological conditions of cells isolated from saliva.

[0099] In some embodiments, the method described in the present disclosure is to detect multiple elements in trace amounts of single cells, and to analyze the dynamic or kinetic parameters of element-specific ICP-MS signal spikes in a particular cell type, and the correlation of one element to other elements at the single cell level in the same biological sample to be detected. In some embodiments, the present disclosure provides a method for assessing the bioavailability of a particular element in a particular cell type, as well as the functional quality of a cell, which comprises, consists essentially of, or even consists of detecting the kinetic or dynamic properties of the ICP-MS single signal spike of a particular element in a particular cell type, such as the dwell time, the peak time of the single signal spike, its ratio to the dwell time, the peak value of the element signal, and the constants and areas under and before and after the peak of the signal spike. See, for example, Figures 1-4 for bioavailability, and Figure 5 for kinetic parameters.

[0100] In some embodiments, the objective of the present disclosure is to solve the technical problem of how to determine the content of multiple elements in a trace amount of cells or even in a single cell, and how to evaluate the functional quality of the cell. In some embodiments, the method of the present disclosure can be applied to determine the composition of multiple elements in a trace amount of cells or even in a single cell, and the determined composition can be used as a reference parameter for evaluating the functional quality of the cell. In some embodiments, the method of the present disclosure can be used to analyze the signal dynamic characteristics of a specific element in a single cell, and the resulting characteristics can be used as a reference parameter for evaluating the functional quality of the cell. In some embodiments, the present disclosure provides a method for evaluating human sperm function. The method comprises, or consists essentially of, or even consists of detecting multiple trace elements in a trace amount of a single cell.

[0101] In some embodiments, the methods disclosed herein assess the quality of culture media and measure the content (eg, absolute amount or concentration) of essential elements and toxic heavy metals.

[0102] In some embodiments, the disclosure herein develops software to analyze elemental bioavailability and dynamic or kinetic properties of element-specific ICP-MS signal spikes in single cells, see Examples.

[0103] In some embodiments, the disclosed method is applicable to all morphologies and types of cells, greatly expanding the scope of application of sc-ICP-MS. In some embodiments, the disclosed method provides a method for detecting multiple trace elements in a single cell at a minute amount, and its use in evaluating cells of heterogeneous morphologies and types. In some embodiments, the disclosed method provides a method for evaluating the functional quality of cells, and its use in evaluating cells of various morphologies and types. In some embodiments, the cells are any species, such as whole somatic cells, gamete cells, prokaryotic cells, or cells isolated from semen, reproductive tract fluid, follicular fluid, blood, urine, saliva, or feces. In some embodiments, the disclosed method uses sperm in human semen as a model to test the feasibility of the disclosed method, and then tests cells isolated from other human body fluids.

[0104] In some embodiments, the methods of the present disclosure can be applied to the following levels: (1) Cellular level: As an example, the methods disclosed herein are used to detect changes in element content in relation to changes in element concentration in the culture medium by adding different concentrations of trace elements to cultured cells; as an additional or alternative example, proteins known to bind to specific elements are overexpressed, knocked down, or knocked out in cells (e.g., gamma-glutamyl carboxylase (GGCX), matrix Gla protein (MGP), occludin (OCLN), TRPV6, TMEM16A, lipocalin (LCN), etc.), and the methods described herein are used to detect differences in elements in cells with or without the addition of the elements. Furthermore, the results can be verified by detecting changes in other elements. (2) Animal level: As an example, using an animal model of infertility or subfertility with known elemental homeostasis disorders, the methods disclosed herein are used to detect fertility of epididymal sperm and / or isolated epithelial cells from infertile animals or animals with low fertility with elemental homeostasis disorders, and as additional or alternative examples, the methods disclosed herein are (a) determining the differences between various elemental properties of epididymal sperm from infertile animals and the changes in various elemental properties of sperm during different capacitation periods, (b) determining the relationship between various elements and known elements when supplementing various elements in the medium in which epididymal sperm from animals that are infertile or animals with low fertility are cultured, or (c) determining whether supplementing various elements orally or by injection improves sperm quality, optionally using CASA, IVF, ICSI, etc., or improving the fertility of the animal by naturally mating the animal with a healthy female. (3) Clinical level: As an example, the method disclosed herein is to analyze sperm collected from clinically healthy men and sperm collected from representative infertile men (e.g., patients with asthenozoospermia or idiopathic infertility, or sperm after cryo-resuscitation, etc.); as an additional or alternative example, the method disclosed herein is to (a) establish the relationship between the content of various elements in sperm and seminal plasma; (b) screen various elements, compare the elemental composition in a known sperm culture medium, and adjust the concentration of various elements in the sperm culture medium to improve the sperm culture medium; or (c) screen various elements in the sperm culture medium to determine which elements or their concentrations improve sperm quality, optionally assessed using CASA, IVF, ICSI, etc.

[0105] Methods and Uses In one aspect, a method is provided for detecting infertile sperm in a sample, the method comprising, consisting essentially of, or even consisting of detecting a concentration of at least one metal selected from the group of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni) outside a predetermined range in sperm using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS). In one embodiment, the at least one metal is selected from sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), or nickel (Ni). In one embodiment, the at least one metal is sodium (Na). In one embodiment, the at least one metal is potassium (K). In one embodiment, the at least one metal is magnesium (Mg). In one embodiment, the at least one metal is zinc (Zn). In one embodiment, the at least one metal is iron (Fe). In one embodiment, the at least one metal is copper (Cu). In one embodiment, the at least one metal is selenium (Se). In one embodiment, the at least one metal is cobalt (Co). In one embodiment, the at least one metal is chromium (Cr). In one embodiment, the at least one metal is cadmium (Cd). In one embodiment, the at least one metal is manganese (Mn). In one embodiment, the at least one metal is arsenic (As). In one embodiment, the at least one metal is mercury (Hg). In one embodiment, the at least one metal is lead (Pb). In one embodiment, the at least one metal is silver (Ag). In one embodiment, the at least one metal is aluminum (Al). In one embodiment, the at least one metal is nickel (Ni).

[0106] In some embodiments, the sperm are capacitated. In some embodiments, the sperm are non-capacitated.

[0107] In some embodiments, the predetermined range corresponds to a concentration of the metal detected in sperm from a population of fertile subjects.

[0108] In some embodiments, the predetermined range of Na concentration is about 5 attograms (ag) to about 50,000 ag for uncapacitated sperm and about 25 ag to about 50,000 ag for capacitated sperm. In some embodiments, the predetermined range of K concentration is about 50 ag to about 50,000 ag for uncapacitated sperm and about 280 ag to about 50,000 ag for capacitated sperm. In some embodiments, the predetermined range of Ca concentration is about 200 ag to about 50,000 ag for uncapacitated sperm and about 700 ag to about 20,500 ag for capacitated sperm. In some embodiments, the predetermined range of Mg concentration is about 8 ag to about 50,000 ag for uncapacitated sperm and about 75 ag to about 15,100 ag for capacitated sperm. In some embodiments, the predetermined range of Zn concentration is about 5 ag to about 50,000 ag for uncapacitated sperm and about 20 ag to about 50,000 ag for capacitated sperm. In some embodiments, the predetermined range of Fe concentration is about 5 ag to about 50,000 ag for uncapacitated sperm and about 13 ag to about 50,000 ag for capacitated sperm. In some embodiments, the predetermined range of Al concentration is about 3 ag to about 50,000 ag for uncapacitated sperm and about 6 ag to about 50000 ag for capacitated sperm (e.g., about 6 ag to about 46700 ag). In some embodiments, the predetermined range of Se concentration is about 59 ag to about 50,000 ag for uncapacitated sperm and about 62 ag to about 45,810 ag for capacitated sperm. In some embodiments, the predetermined range of Co concentration is from about 3 ag to about 3,700 ag for uncapacitated sperm and from about 9 ag to about 20,200 ag for capacitated sperm.In some embodiments, the predetermined range of Cu concentration is about 9 ng to about 50,000 ag for uncapacitated sperm and about 9 ng to about 37,590 ag for capacitated sperm. In some embodiments, the predetermined range of Cr concentration is about 4 ng to about 50,000 ag for uncapacitated sperm and about 5 ag to about 46,700 ag for capacitated sperm. In some embodiments, the predetermined range of Mn concentration is about 2 ag to about 50,000 ag for uncapacitated sperm and about 7 ag to about 32,610 ag for capacitated sperm.

[0109] In some embodiments, the mean mass As concentration corresponds to about 60 ag per cell per sperm, either capacitated or uncapacitated, the predetermined range of mean mass Ag concentration corresponds to about 800 ag per cell per sperm, either capacitated or uncapacitated, the predetermined range of mean mass Cd concentration corresponds to about 510 ag per cell per sperm, either capacitated or uncapacitated, the mean mass Hg concentration corresponds to about 5400 ag per cell per sperm, either capacitated or uncapacitated, the predetermined range of mean mass Pb concentration corresponds to about 1610 ag per cell per sperm, either capacitated or uncapacitated, and the mean mass Ni concentration corresponds to about 2570 ag per cell per sperm, either capacitated or uncapacitated. As one of ordinary skill in the art will appreciate, corresponding predetermined ranges may refer to the same ranges, but are determined or calculated using parameters other than the ag of metal per sperm, such as the concentration of the metal in the sperm, and therefore such corresponding predetermined ranges are also included within the disclosure herein.

[0110] In one aspect, a method is provided for detecting infertile sperm in a sample, the method comprising, consisting essentially of, or even consisting of detecting a dynamic or kinetic parameter of a signal spike of at least one metal selected from the group of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni) outside a predetermined range of sperm using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS).

[0111] In some embodiments, the parameters correspond to dynamic or kinetic parameters detected in sperm from a population of fertile subjects.

[0112] In some embodiments, the dynamic or kinetic parameters of the spike are selected from dwell time, dwell time before the peak, dwell time after the peak, peak time, ratio between peak time and dwell time, rising tau constant before the peak, dynamic area before the peak, tailing tau constant after the peak, dynamic area after the peak, or any combination thereof. In some embodiments, the detected dynamic or kinetic parameters of the spike include, consist essentially of, or even consist of any one or more of the following: (a) residence time of the Fe spike of about 1.4 to about 7.9 ms for uncapacitated sperm and about 1.5 to about 6.7 ms for capacitated sperm; (b) tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.81 ms for uncapacitated sperm and about 0.18 to about 0.90 ms for capacitated sperm; (c) rising tau constant before the peak of the Fe spike of about -0.35 ms or less for uncapacitated sperm and about -0.80 ms for capacitated sperm; (d) residence time of the Cu spike of about 1.5 ms or less for uncapacitated and capacitated sperm; (e) tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.81 ms for uncapacitated sperm and about 0.18 to about 0.90 ms for capacitated sperm; ) a rising tau constant before the peak of the Cu spike of about -0.2 ms or less for uncapacitated sperm, and a rising tau constant before the peak of the Cu spike of about -0.6 ms or less for capacitated sperm, (f) a tailing tau constant after the peak of the Cu spike of about 0.15 ms or less for uncapacitated sperm, and a tailing tau constant after the peak of the Cu spike of about 0.2 ms or less for capacitated sperm, (g) a dwell time of the Zn spike of about 2.1 ms or less for uncapacitated sperm, and a dwell time of the Zn spike of about 1.2 ms or less for capacitated sperm, (h) a rising tau constant before the peak of the Zn spike of about -0.25 ms or less for uncapacitated sperm, and a tailing tau constant after the peak of the Cu spike of about -0.(i) a rising tau constant before the peak of the Zn spike of 20 ms or less, (ii) a tailing tau constant after the peak of the Zn spike of about 1.15 ms or less for uncapacitated sperm, and a tailing tau constant after the peak of the Zn spike of about 0.25 ms or less for capacitated sperm, (iii) a residence time of the Cr spike of about 3.25 ms or less for uncapacitated sperm, and a tailing tau constant before the peak of the Cr spike of about -0.45 ms or less for uncapacitated sperm. (k) for uncapacitated sperm, a tailing tau constant after the peak of the Cr spike of about 0.2 to about 0.5 ms; (l) for uncapacitated sperm, a residence time of the Se spike of about 1.5 ms or less; (m) for uncapacitated sperm, a rising tau constant before the peak of the Se spike of about -0.15 ms or less; and (n) for uncapacitated sperm, a tailing tau constant after the peak of the Se spike of about 0.25 ms or less.

[0113] In one aspect, a method for detecting infertile sperm in a sample is provided, the method comprising, or consisting essentially of, or even consisting of: (i) contacting a first population of sperm from a subject with a human tubal fluid (HTF) buffer and optionally centrifuging the first population, and (ii) using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) to detect a concentration of at least one metal selected from the group of potassium (K), calcium (Ca), magnesium (Mg), mercury (Hg), silver (Ag), and aluminum (Al) in the first population of sperm after the contacting step that is equal to or lower than the concentration present in a second population of sperm that is not contacted with the HTF buffer, or (iii) using sc-ICP-MS to detect a concentration of selenium (Se) in the first population of sperm after the contacting step that is equal to or higher than the concentration present in the second population of sperm that is not contacted with the HTF buffer, or (iv) both (ii) and (iii).

[0114] In some embodiments, the sample is obtained from a subject, hi some embodiments, the subject has or is suspected of having idiopathic infertility, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia.

[0115] In some embodiments, the methods disclosed herein further comprise treating the subject with an infertility therapy. In some embodiments, the infertility therapy comprises, consists essentially of, or even consists of administering an element, the level of which is detected in the subject's sample compared to the control. In some embodiments, the concentration of at least one metal in sperm is lower than a predetermined range, and the infertility therapy comprises treatment with at least one metal. Additionally or alternatively, the infertility therapy comprises, consists essentially of, or even consists of administering an agent, such as a small molecule or protein, that binds to the element, the level of which is detected in the subject's sample compared to the control. In some embodiments, the concentration of the metal is detected in sperm below a predetermined range, and the infertility therapy comprises at least one metal. In some embodiments, the subject is treated with an infertility therapy or infertility treatment.

[0116] In some embodiments, more than one of the metals is detected.

[0117] In some embodiments, the sample is diluted with a buffer. In some embodiments, the sample is diluted with a buffer prior to the detecting step. In some embodiments, the spermatozoa are diluted to 3×10 6 or less prior to detection by sc-ICP-MS. 6 The sperm are diluted to a concentration of 0.01 sperm / ml or less. Additionally or alternatively, the sperm are diluted 10-fold or more prior to detection by sc-ICP-MS. In some embodiments, the sperm are diluted using any one of the following buffers: HTF buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane (tris) buffer, or bis-trismethane (bis-tris) buffer.

[0118] In some embodiments, the sperm are centrifuged to remove seminal plasma before dilution.

[0119] In some embodiments the sample comprises, or consists essentially of, or even consists of semen, hi further embodiments the semen is liquefied, or fixed, or capacitated, or cryopreserved, or liquefied and fixed, or liquefied and capacitated, or liquefied and cryopreserved, or fixed and capacitated, or fixed and cryopreserved, or capacitated and cryopreserved, or liquefied, fixed and capacitated, or liquefied, fixed and cryopreserved, or liquefied, capacitated and cryopreserved, or liquefied, fixed, capacitated and cryopreserved.

[0120] In some embodiments, the methods disclosed herein further comprise performing computer assisted sperm analysis (CASA) on the sperm.

[0121] In some embodiments, the methods disclosed herein further comprise purifying sperm having (i) at least one metal concentration, or (ii) a dynamic or kinetic parameter, or both (i) and (ii), within a predetermined range to obtain functional sperm.

[0122] In some embodiments, the methods disclosed herein further comprise fertilizing an egg with the purified functional sperm, e.g., via in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

[0123] In some embodiments, the methods disclosed herein detect all somatic, gametic, and prokaryotic cells, regardless of their species.

[0124] In some embodiments, the detection concentration of cells is 3×10 6 / ml.

[0125] In some embodiments, more than two elements are detected.

[0126] In some embodiments, the dynamic or kinetic parameters (also referred to herein as dynamic or kinetic properties or dynamic or kinetic features) of sc-ICP-MS signal spikes of elements in cells comprise, consist essentially of, or even consist of any one or more of the following: dwell time, peak time of a single signal spike, ratio of peak time to dwell time, area of ​​the peak, pre-peak kinetic constant, pre-peak area, post-peak kinetic constant, or post-peak area. See, e.g., FIG. 5.

[0127] In some embodiments, the disclosure herein provides a correlation between elemental bioavailability and calcium content at the single cell level of the same biological sample, for example, correlation between elemental bioavailability and calcium content in sperm cells (see, e.g., Figures 14-17), wild type somatic cells, pathological somatic cells such as cancer cells (see, e.g., Figures 18-21).

[0128] In some embodiments, the disclosure herein provides a profile of the frequency distribution of the average mass of various elements detected in single human sperm cells in four physiological states (see, e.g., FIG. 22 ), and the identification of subpopulations of essential elements in single cells of the same biological sample, e.g., a profile of elemental bioavailability with iron (Fe) content in sperm cells (see, e.g., FIG. 23 ).

[0129] In some embodiments, unique single-cell elemental profiles can be discovered in normal and abnormal (e.g., oligoasthenospermia) human sperm.

[0130] In some embodiments, single cell elemental profiles of essential elements, including Mg, Zn, Fe, Cu, Cr, and Mn, are associated with risk of abnormal sperm function, such as risk of oligoasthenozoospermia.

[0131] In some embodiments, the single-cell element-specific kinetic characteristics of ICP-MS signals in human sperm differ from those of somatic cells, such as cancer cells.

[0132] In some embodiments, the disclosure herein provides a method for the analysis of a number of different functional algorithms, including but not limited to: 1) exponential alpha, 2) exponential cumulative probability, 3) exponential log probability, 4) exponential power, 5) exponential probability, 6) exponential product, 6) exponential slope baseline, 7) exponential normal, 8) exponential weighting, 9) exponential weighting / constraint, 10) Guassian, 11) binomial, 12) polynomial, 13) Boltzmann charging voltage, 14) Boltzmann shift, 15) Boltzmann normal, 16) Boltzmann Z-delta (ascending), 17) Boltzmann Z-delta (descending), 18) skewness, 19) kurtosis, 20) nonlinear regression (curve fitting). , 21) Simple linear regression, 22) Simple logistic regression, 23) Fit splines / smoothing, 24) Smooth, differential, or integral curves, 25) Area under the curve, 26) Standard curve interpolation, 27) t-tests (and nonparametric tests), 28) One-way ANOVA (and nonparametric or mixed models), 29) One-sample t and Wilconxon tests, 30) Frequency distributions, 31) Outliner identification, 32) Bland-Altman comparison, 33) Two-way ANOVA (or mixed models), 34) Three-way ANOVA (or mixed models), 35) Multiple t-tests (and nonparametric tests), 36) Chi-square (and Fisher's exact) test, 36) Fraction The present invention provides correlation relationships of single-cell element-specific kinetic properties with specific elements or combinations of two or more elements related to analytical results by applying AI (artificial intelligence) for matrix algorithm calculations, including 3) of Total, 37) correlation matrix, 38) multiple linear regression, 39) multiple logistic regression, 39) principle component analysis, 40) nested t-test, 41) nested one-way ANOVA, etc.

[0133] In some embodiments, the disclosure herein provides correlations between single-cell element-specific kinetic characteristics and a subject's sperm functional quality or health status, such as metabolic syndrome and related health conditions.

[0134] In some embodiments, the present disclosure relates to a method and use for detecting multiple trace elements in trace cells, which belongs to the technical field of medical detection. The present disclosure includes two parts: detection of multiple trace elements and signal kinetic analysis. Conventional single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) method can be used. Using a single cell nebulizer, cell suspension is aspirated into droplets, each containing a single cell, into a single cell spray chamber, and the bioavailability of elements and their kinetic characteristics of ICP-MS signal spikes are analyzed at the single cell level using a high-speed digitization software module, and standards are prepared and detected using the same method. The kinetic characteristics of specific elements in specific cell types are then analyzed. The present disclosure can be applied to all somatic cells, gamete cells, prokaryotic cells, and human cancer cells, and determines the content of multiple elements at the trace cell or even single cell level, thereby providing reference parameters for evaluating the functional quality of cells. In some embodiments, the cells include, but are not limited to, cells isolated from semen, reproductive tract fluid, follicular fluid, blood, urine, saliva, and feces.

[0135] The subject matter of the present disclosure relates to a method for the preparation of a single cell, e.g., a microorganism isolated from a biological sample from a subject (e.g., 1×10 6 The present disclosure is directed to a method for aiding in the diagnosis, prognosis, monitoring and evaluation of disease or other medical conditions in a subject by detecting to determine element availability (content of more than two elements) in sperm or somatic cells (at cell densities or concentrations less than 1×10 / ml). The method is to detect multiple trace and essential elements in trace cells and analyze the signal kinetics and dynamics of the elements in the sample. The main steps of the multiple trace element detection part of the present disclosure are obtaining a cell-containing fluid, with or without fixation, and centrifuging the supernatant and diluting with diluent to obtain a detection concentration of 1×10 6The method includes measuring the concentration of the element in the sample in a single sample, measuring the concentration of the element in the single ... in a single sample, measuring the concentration of the element in the single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample in a single sample

[0136] In some embodiments, the methods disclosed herein include obtaining somatic cells by centrifugation of a sample, removing the supernatant, and centrifuging the cells in a diluent at 3×10 6 The method includes, or consists essentially of, or even consists of, diluting the cell suspension to a detection concentration of less than 1000 μg / ml, and loading the diluted cells into a conventional single cell ICP-MS (sc-ICP-MS) instrument for detection. In some embodiments, the detection method uses conventional single cell ICP-MS (sc-ICP-MS). Using a nebulizer, the cell suspension is aspirated into small droplets, each containing a single cell, into a single cell spray chamber, detecting standards using the same procedure and generating standard curves of elements, and using high-speed analysis digitizing software as provided by the manufacturer of the sc-ICP-MS machine to detect elemental spike signals of each single cell in the small droplets, and analyzing the bioavailability of multiple trace elements and element-specific spike signal characteristics analysis at the single cell level.

[0137] In some embodiments, to solve the problems discussed herein, what is disclosed herein is a method for detecting multiple trace elements in a trace amount of cells, the method comprising, consisting essentially of, or even consisting of the following steps: Step 1: Obtain somatic cells, centrifuge the cells, remove the supernatant, and solubilize the centrifuged cells in diluent to 3 × 10 6 The cells are suspended to a detection concentration of less than 1 / ml and the suspended cells are loaded into an sc-ICP-MS instrument for detection. Step 2: The detection method is carried out using conventional sc-ICP-MS techniques. A single-cell nebulizer is used to aspirate the cell suspension into droplets in a single-cell spray chamber, each of which constitutes a single cell, and a rapid analysis digitization software module is used to analyze multiple trace elements at the single-cell level. Step 3: Using the same procedure in step 2, detect a series of standard solutions with known concentrations of elements and plot a standard curve. Step 4: Determine the unique elemental ICP-MS signal characteristics of a particular cell type using the method, which includes evaluating the sc-ICP-MS signal kinetics characteristics of a particular element in a particular cell selected from dwell time, peak time of a single signal spike, ratio of pre-peak or post-peak time of the spike relative to dwell time, dynamic kinetic constant or pre-peak area of ​​the spike, dynamic kinetic constant or post-peak area of ​​the spike, etc. Step 5: Assess the functional quality of the cells.

[0138] In some embodiments, the present disclosure provides a method for detecting multiple trace elements in human sperm cells, the method comprising, consisting essentially of, or even consisting of the following steps: Step 1: Sample preparation: Liquefy fresh human semen according to conventional semen preparation methods, centrifuge the liquefied semen to separate seminal plasma and sperm, resuspend the sperm in 4% PFA, fix the sperm in 4% PFA for 15 minutes, remove the fixative by washing, and perform the following steps with the sperm or store the sperm at 4°C for later use. Step 2: Dilute the sperm to 3 x 10 6 Dilute to a detection concentration of less than 1 / ml. Step 3: Load the sample into the sc-ICP-MS instrument for detection. A specific single-cell nebulizer and spray chamber were installed and calibrated with an adjustment solution (2% v / v nitric acid containing 10 μg / L Li, Be, Mg, Fe, In, Ce, Pb and U) to determine the appropriate instrument detection parameters; gold particle standards were used to determine the single-cell transmission efficiency, which is generally in the range of 40%-60%; standard solutions of different concentrations of different elements were prepared and standard curves were plotted; all standard solutions were prepared in ultrapure water; to eliminate polyatomic interferences and obtain a high signal-to-noise ratio, measurements were performed in dynamic reaction cell (DRC) mode using ammonia as a reactant gas for the detection of K, Ca, Cr, and Fe in single cells, or using oxygen as a reactant gas for the detection of As and Se in single cells by detecting the oxidation reaction products of AsO and SeO as analytes; other elements were measured in standard mode; the sample spike signal was sampled for 50 s with a dwell time of 50 μs (i.e., digitization frequency at 50 MHz); the total sample volume consumed by detecting 18 elements was approximately 400 μL. Step 4: Determine the unique elemental ICP-MS signal characteristics of a particular cell type using the method, which includes evaluating sc-ICP-MS signal kinetics characteristics of a particular element in a particular cell selected from dwell time, peak time of a single signal spike, ratio of pre-peak or post-peak time of the spike relative to dwell time, dynamic kinetic constant or pre-peak area of ​​the spike, and dynamic kinetic constant or post-peak area of ​​the spike. Step 5: Assess the functional quality of the cells.

[0139] kit Additionally, kits are provided that comprise, consist essentially of, or even consist of buffers and instructions for practicing the methods disclosed herein.

[0140] In some embodiments, the kit includes a sterile container containing the infertility therapy or buffer, or both, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0141] In some embodiments, the instructions generally include information regarding the use of the buffer and optional infertility therapy for the methods disclosed herein. In other embodiments, the instructions include at least one of the following: description of the buffer, description of the optional infertility therapy, dosage schedule and administration of the optional infertility therapy, precautions, warnings, indications, counterindications, overdose information, adverse reactions, animal pharmacology, clinical trials, or bibliographic references. The instructions may be printed directly on the container (if present), printed as a label affixed to the container, or printed as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0142] A device that can deliver the kit components via an administration route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices. In some embodiments, the infertility therapy of the present technology may be provided in the form of a pre-filled syringe or auto-injection pen that contains a sterile liquid formulation or a lyophilized formulation (e.g., Kivitz et al., 2006, Clin. Ther. 28:1619-29).

[0143] The kit components may be packaged together or separated into two or more containers. In some embodiments, the container may be a vial containing a sterile, lyophilized formulation of the infertility therapy suitable for reconstitution. The kit may also include one or more buffers suitable for reconstitution or dilution, or both reconstitution and dilution of other reagents. Other containers that may be used include, but are not limited to, pouches, trays, boxes, tubes, and the like. The kit components may be packaged and maintained aseptically within the container.

[0144] The following examples are included to demonstrate some embodiments of the present disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES

[0145] Example 1: Method and application for detecting multiple trace elements in trace cells In order to make the present disclosure easier to understand, some embodiments and accompanying drawings are described in detail as follows: The present disclosure provides a method for detecting multiple trace elements in a trace amount of cells, comprising the steps of: Step 1: Obtain gamete cells and / or somatic cells, centrifuge the cells, remove the supernatant, and soak the centrifuged cells in diluent to 3 × 10 6 The cells are suspended to a detection concentration of less than 1 / ml and the suspended cells are loaded into an sc-ICP-MS instrument for detection. Step 2: The detection method is carried out using conventional sc-ICP-MS techniques. A single-cell nebulizer is used to aspirate the cell suspension into droplets in a single-cell spray chamber, each of which constitutes a single cell, and a rapid analysis digitization software module is used to analyze multiple trace elements at the single-cell level. Step 3: Using the same procedure in step 2, detect a series of standard solutions with known concentrations of elements and plot a standard curve. Step 4: Determine the unique elemental ICP-MS signal characteristics of a particular cell type using the method, which includes evaluating the sc-ICP-MS signal kinetics characteristics of a particular element in a particular cell selected from dwell time, peak time of a single signal spike, ratio of pre-peak or post-peak time of the spike relative to dwell time, dynamic kinetic constant or pre-peak area of ​​the spike, dynamic kinetic constant or post-peak area of ​​the spike, etc. Step 5: Assess the functional quality of the cells.

[0146] As shown in Figures 1-5, a method is provided for detecting multiple trace elements in a trace amount of cells for use in evaluating human sperm cells, the method comprising, or consisting essentially of, or even consisting of: Step 1: Sample preparation: Liquefy fresh human semen according to conventional semen preparation methods, centrifuge the liquefied semen to separate seminal plasma and sperm, resuspend the sperm in 4% paraformaldehyde (PFA), fix the sperm in 4% PFA for 15 minutes, remove the fixative by washing, and use the fixed sperm to perform the following steps or store the fixed sperm at 4°C for later use. Step 2: Dilute the sperm to 3 x 10 6 Dilute to a detection concentration of less than 1 / ml. Step 3: Load the sample into the sc-ICP-MS instrument for detection. Set up a specific single-cell nebulizer and spray chamber, adjust with an adjustment solution (2% v / v nitric acid containing 10 μg / L Li, Be, Mg, Fe, In, Ce, Pb and U) to determine the appropriate instrument detection parameters, use gold particle standards (e.g., 50 nm gold particles) to determine the single-cell penetration efficiency, which was generally in the range of 40%-60%, prepare standard solutions with different concentrations of different elements (e.g., Na, K, Ca, Mg, Zn, Fe, Cu, Se, Co, Cr, Cd, Mn, As, Hg, Pb, Ag, Al, Ni), plot the standard curve, and the concentrations of the standard solutions of Hg are 0.5 parts per billion (ppb), 1 ppb, and 2 ppb, and the concentrations of the standard solutions of Ca are 50 ppb, 100 ppb, and 200 ppb. , the concentrations of standard solutions of other elements were 5 ppb, 10 ppb, and 20 ppb, and all standard solutions were prepared in ultrapure water. To eliminate polyatomic interferences and obtain a high signal-to-noise ratio, measurements were performed in dynamic reaction cell (DRC) mode using ammonia as a reactant gas for the detection of K, Ca, Cr, and Fe in single cells, or oxygen as a reactant gas for the detection of As and Se in single cells by detecting the oxidation reaction products of AsO and SeO as analytes. Other elements were measured in standard mode, and the sample spike signal was sampled for 50 seconds with a dwell time of 50 μs (i.e., digitization frequency at 50 MHz). The total sample volume consumed by detecting 18 elements was about 400 μL. The normal working conditions of the sc-ICP-MS are shown in Table 1 below. [Table 1]

[0147] There is still a lack of methods for single sperm ion spectrum analysis in clinics. Moreover, the ion content in sperm is also an important factor for evaluating sperm quality. Therefore, it is important to establish a method for detecting the ion spectrum of individual sperm, provide a diagnostic method for evaluating sperm quality in clinical laboratories and cell function, thereby providing references and values ​​in this regard. Furthermore, the unique characteristics of ICP-MS signals of specific elements can be distinguished in single sperm cells, including the residence time of different elements in single sperm, the peak time of a single signal and its ratio to the residence time, the kinetic constant and area before the peak, and the kinetic constant and area after the peak. These characteristics of ICP-MS signal dynamics have important biological significance and far-reaching implications related to pathological state evaluation, and therefore have clinical application implications.

[0148] As shown in Figure 1, the quantitative analysis of multiple elements in single human sperm in different samples is provided.By using the detection method disclosed herein, the difference of multiple elements in different samples is detected.The lowest sensitivity for the detection of multiple elements reaches the concentration of attogram (ag) per cell (see Figure 1).

[0149] As shown in Figure 2, some samples are provided with relatively high content of essential trace elements in single human sperm. The detection method disclosed herein detects the difference in the content of essential trace elements in different samples. These findings can be used as a reference, and suggestions for element supplementation can be made to subjects who are deficient in these elements.

[0150] As shown in Figure 3, some examples of samples with relatively high contents of toxic elements in single human sperm are provided. These subjects with relatively high levels of toxic elements can be treated for clinical detoxification.

[0151] Examples of some samples with relatively high contents of other elements in single human sperm are provided, as shown in Figure 4. These reference parameters have clinical implications.

[0152] As shown in Figure 5, the sc-ICP-MS single signal kinetics of a particular element is provided. The figure shows the cell-specific element-specific characteristics, including the dwell time, the peak time of the single signal spike and its ratio relative to the dwell time, the kinetic constant of the spike or the area before the peak, and the kinetic constant of the spike or the area after the peak, based on the sc-ICP-MS signal kinetics of iron and copper elements in a single human sperm cell of the same sample as an example.

[0153] Although different cells contain different elemental contents, specific elements in different cells show unique ICP-MS signals. Thus, such parameters can distinguish the specific characteristics of different cells. For example, the time and constants of sc-ICP-MS signal-related characteristics of different elements are distinguishable in cultured mouse epididymal epithelial DC2 cells, human embryonic kidney 293T cells, human cervical cancer HeLa cells, and human gastric cancer SNU-1 cells. These ICP-MS signal dynamics characteristics have important biological significance and far-reaching implications related to pathological state evaluation, and therefore have implications for clinical applications.

[0154] FIG. 6 shows the signal characteristics of essential macronutrients (calcium and magnesium) in different cell types.

[0155] FIG. 7 shows the signal characteristics of essential trace elements in human sperm, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), cobalt (Co), and selenium (Se).

[0156] Figure 8 shows the signal characteristics of essential trace elements in mouse epididymal epithelial DC2 cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), cobalt (Co), and selenium (Se).

[0157] FIG. 9 shows the signal characteristics of essential trace elements in human embryonic kidney 293T cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), chromium (Cr), selenium (Se), etc.

[0158] FIG. 10 shows the signal characteristics of essential trace elements in human cervical cancer HeLa cells, including zinc (Zn), iron (Fe), manganese (Mn), and chromium (Cr).

[0159] FIG. 11 shows the signal characteristics of essential trace elements in human gastric cancer SNU-1 cells, including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and chromium (Cr).

[0160] Figure 12 provides the signal characteristics of toxic elements in different cell types. Figure 12A shows human sperm. Figure 12B shows mouse epididymal epithelial DC2 cells. Figure 12C shows human embryonic kidney 293T cells. Figure 12D shows human cervical cancer Hela cells. Figure 12E shows human gastric cancer SNU-1 cells. The signal values ​​and kinetic characteristics of these toxic elements have biotoxicological and pathological significance and can be used for clinical diagnosis.

[0161] Example 2: Use of Elemental Signature Analysis in the Diagnosis and Prognosis of Medical Diseases and Conditions and Methods Therefor Elementomic characterization of normal and dysfunctional human sperm using single-cell ICP-MS: Today, sperm dysfunction is the leading cause of male infertility, but the diagnostic failure rate still accounts for 30% to 70% of clinical cases, most of which are still classified as idiopathic. This is in part because traditional assessment methods of sperm function cannot fully meet clinical needs. In an attempt to determine whether elemental bioavailability profiles in single human sperm could be an approach to reveal functional associations with male factor infertility, single-cell inductively coupled plasma mass spectrometry (sc-ICP-MS) was utilized. To this end, elementomic bioavailability profiles were characterized for human normal sperm and dysfunctional sperm with oligoasthenospermia. It was found that not only elementomic profiles but also the intrinsic signatures of certain elements in human sperm are associated with the risk of oligoasthenospermia. Without wishing to be bound by this theory, the single-cell elementomic characterization approach can be used to analyze sperm function and other health conditions related to male fertility.

[0162] Human sperm sample preparation: All included human studies were approved by the Medical Ethics Review Board of Shanghai Institute for Biomedical and Pharmaceutical Technologies (formerly "Shanghai Institute of Planned Parenthood Research"), and the Research Ethics Board of ShanghaiTech University. Semen samples were collected using the WHO-introduced method. With the patient's consent, the remaining sperm samples were collected without interfering with clinical use. A total of 68 specimens were collected from hospitals including the Reproductive Medicine Center of Zhongshan Hospital affiliated with Fudan University, Shanghai Jiai Genetics and IVF Institute. Within these tested samples, 34 of them were original semen samples and 34 were capacitated sperm samples for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) assays. Nine of them had both original semen samples and were capacitated. All these samples were collected by masturbation after the recommended abstinence of 3-5 days from sexual intercourse or masturbation. Sperm count and motility of collected semen were measured by computer-assisted sperm analysis (CASA) (37°C), and sperm samples for IVF / ICSI were processed by density gradient according to the WHO laboratory manual for the examination and processing of human semen at the sampling site. Relevant information is shown in Table 2.

[0163] Samples were collected from four physiological states of sperm cells at 1 × 10 per ml. 6The sperm were prepared to a sperm density of less than 100 mg / mL. The sperm were classified as follows: 1) normal semen that had not undergone capacitation (normal sperm, uncapacitated), 2) normal viable motile sperm that had been processed by standard procedures of density gradient centrifugation in commercially available HTF-HEPES buffer for sperm to undergo capacitation during centrifugation (normal viable DGC-capacitated sperm), 3) asthenozoospermia or oligoasthenozoospermia that had not undergone capacitation, and 4) viable DGC-capacitated sperm, but oligoasthenozoospermia (capacitated oligoastheno). Oligoasthenozoospermia was classified according to the lower limit criteria for semen characteristics described in the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen.

[0164] Normal samples were 15 x 10 per ml. 6 and asthenozoospermia samples had a concentration of >15 × 10 per ml in the original semen. 6 was identified at concentrations below 0.05, while oligozoospermia was in samples with sperm motility below 40% (PR+NP%) (Agarwal et al., 2021, and WHO, 2010, WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th Edition).

[0165] [Table 2]

[0166] Aliquots of collected sperm semen in 2 ml Eppendorf tubes were then placed in Styrofoam-protected containers at room temperature (RT) within 2 h and sent to the sc-ICP-MS laboratory for subsequent experimental preparation. Sperm were spun down at 500 g for 10 min, fixed in 4% PFA for 15 min at room temperature, subsequently washed three times with pure water, and then diluted to the desired concentration for sc-ICP-MS measurements in Milli-Q water. All sperm samples were collected during autumn or winter. Sperm were immediately aspirated with a 1 ml syringe infusion pump (Harvard Apparatus) into the suction chamber for sc-ICP-MS analysis. For whole cell measurements, cells were digested with concentrated nitric acid for 30 min at 99 °C. The digested solution was then diluted with 2% HNO in Milli-Q water for 1 h. 3 and determined by ICP-MS.

[0167] Culture cell sample preparation: DC2 cell line (one of the mouse epididymal epithelial cell lines, and a gift from Dr. Yong-Lian Zhang's laboratory) was cultured at 33°C, 5% CO in Full-IMDM (Iscove's modified Dulbecco's medium (IMDM) supplemented with 1 nM 5α-dihydrotestosterone and containing 10% (v / v) fetal bovine serum (FBS), penicillin (100 g / mL), and streptomycin (100 g / mL)). 2 Human gastric cancer cell line SNU-1 (a gift from Dr. Shuo Shi, ShanghaiTech University) was cultured at 37°C in 5% CO in Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with streptomycin (100 g / mL), penicillin (100 U / mL), glutamine (2 mM), and 10% (v / v) FBS (complete medium). 2 HEK293T and HeLa cell lines were cultured in a 37°C, 5% CO incubator using complete DMEM (Dulbecco's modified Eagle's medium) containing 10% (v / v) FBS, penicillin (100 g / mL), and streptomycin (100 g / mL). 2The cultured cells were digested into single dispersed cells using TrypLE, washed three times with PBS, spun down at 1000g for 5 min, and either fixed or not fixed in 4% PFA at room temperature for 15 min, followed by washing three times with pure water and removing cell clumps with a 70-mesh sieve to ensure single cell samples, and then diluted with Milli-Q water to the desired concentration for sc-ICP-MS measurements.

[0168] Sample preparation: Fresh human semen was liquefied and centrifuged according to conventional semen preparation methods. Seminal plasma and sperm were then separated, and sperm were resuspended and fixed in 4% PFA for 15 minutes. The fixative was then removed by washing, and sperm samples were either used directly or stored at 4 degrees for later use.

[0169] The sample was diluted with diluent to 3 x 10 6 The detection concentration was less than / ml.

[0170] The samples were then loaded into the sc-ICP-MS instrument for detection, and a specific single-cell nebulizer and spray chamber were installed. A calibration solution (2% v / v nitric acid containing 10 μg / L Li, Be, Mg, Fe, In, Ce, Pb, and U) was used to determine the appropriate instrument detection parameters. Gold particle standards (e.g., 50 nm gold particles) were used to determine the single-cell penetration efficiency, which generally ranged from 40% to 60%. Standard solutions of different elements (e.g., Na, K, Ca, Mg, Zn, Fe, Cu, Se, Co, Cr, Cd, Mn, As, Hg, Pb, Ag, Al, Ni) at different concentrations were prepared. Standard curves were plotted accordingly. The concentrations of the standard solutions of Hg were 0.5 parts per billion (ppb), 1 ppb, and 2 ppb. The concentrations of the standard solutions of Ca were 50 ppb, 100 ppb, and 200 ppb. The concentrations of standard solutions of other elements were 5 ppb, 10 ppb, and 20 ppb. All standard solutions were prepared in ultrapure water. To eliminate polyatomic interferences and obtain a high signal-to-noise ratio, measurements were performed in dynamic reaction cell (DRC) mode using ammonia as a reactant gas to detect K, Ca, Cr, and Fe in single cells, or oxygen as a reactant gas to detect As and Se in single cells by detecting the oxidation reaction products of AsO and SeO as analytes. Other elements were measured in standard mode. The sample spike signal was sampled for 50 seconds with a dwell time of 50 μs (i.e., digitized at a frequency of 50 MHz), and the total sample volume consumed by detecting 18 elements was about 400 μL. The typical operating conditions of the sc-ICP-MS are shown in Table 3 below. Exemplary results can be found in Figures 1-5. [Table 3] Notes: DRC: Dynamic Response Cells.

[0171] As illustrated in Figures 6-12, the examples show the characteristics of sc-ICP-MS signal kinetics of different elements in single human sperm, including the dwell time of single signals (total peak dwell time), the peak time of single signals during the dwell time, the dwell time before the peak and the rising tau constant before the peak, and the dwell time after the peak and the tailing tau constant after the peak. It was found that not only do different cells contain different elements (bioavailability), but certain elements also have unique ICP-MS signals of special elements in single cells, which can be identified in different cell types. For example, the time and constant of sc-ICP-MS signal related characteristics of different elements can distinguish different cell types, such as cultured mouse epididymal epithelial DC2 cells, human embryonic kidney 293T cells, human cervical cancer HeLa cells, and human gastric cancer SNU-1 cells. The biological significance of these signal kinetic characteristics, the associated pathological significance, and the prospects for clinical applications are under investigation.

[0172] Figures 13-14 provide the characteristics of the average mass profiles of elements in single human sperm from normal and oligoasthenospermia samples.

[0173] When analyzing the elemental average mass data obtained from the ICP-MS readings, it was noticed that the values ​​of some elements changed rapidly between readings. To determine the cause of this change, the elemental content was analyzed in relation to the dilution factor and sperm density of the samples. The results showed that at lower cell densities, the average mass of elements remained relatively stable. 6The mean contents of Na, Ca, Mg and Zn elements correlated significantly and increased with increasing cells when the sperm samples were diluted 10-fold or more, regardless of the original cell concentration. In some embodiments, the results also showed that the mean contents of most elements remained stable when the sperm samples were diluted 10-fold or more, regardless of the original cell concentration. Although no statistical differences were determined at the tested dilution factors of the samples, a trend of higher mean mass was determined for Na, K, Ca, Mg, Zn and Fe elements at the original dilution factor. Further analysis was then performed for these elements at dilution factors of 10-fold or more. Therefore, for statistical analysis, the mean mass of the elements was calculated as 1×10 per ml or less. 6 The cultures were divided into two groups, with lower and higher cell densities, both at 10-fold or greater dilution.

[0174] The results showed that only the average content of Ca was significantly increased when comparing normal sperm diluted from the original semen sample with viable motile sperm from samples treated with a density gradient centrifugation (DGC) procedure in HTF buffer, which also allowed the sperm to undergo capacitation and be collected with good motility (Figure 13A). This result is consistent with the fact that Ca is essential for sperm capacitation and hyperactivation. During these processes, large amounts of calcium ions flow into sperm cells, thus activating them and preparing them for the acrosome reaction and fertilization (Ickowicz et al., 2012, Asian J Androl 14, 816-821, and Navarrete et al., J Cell Physiol, 2015, 230, 1758-1769). Impaired calcium homeostasis in sperm leads to defects in sperm motility and capacitation, and subsequently to male infertility in mice, accompanied by increased resting levels of intracellular calcium in sperm cells (Okunade et al., 2004, J Biol Chem 279, 33742-33750, and Schuh et al., 2004, J Biol Chem 279, 28220-28226).

[0175] The results also showed that in sperm with higher cell density, the mean mass of Zn element was significantly increased under control conditions, but not after the DGC capacitation procedure (Figure 13A). Several other elements also showed a greater mean mass content in sperm with higher density, although there was no statistical difference. Without wishing to be bound by theory, two mechanisms were proposed herein to explain this phenomenon. The first mechanism is that this was due to the matrix background inherited from the original seminal plasma. In support of this mechanism, the fold change in the mean mass of most elements, including the macro-essential elements Na, Ca, and Mg, and the trace-essential elements Zn, Cr, Fe, Cu, and Se, increased at higher cell density (Figure 13C). Furthermore, it has been reported that the cell surface glycocalyx regulates the surrounding sodium homeostasis, thus controlling the permeability and sodium-potassium pump activity in pathophysiological conditions (Korte et al., 2012, Pflugers Arch 463, 269-278). Furthermore, the average mass of K was found to be substantially inversely correlated with sperm Na (Figure 14A). Without wishing to be bound by theory, another mechanism is proposed herein that may be due to ion trapping in the carbohydrate-rich glycocalyx coating on the sperm membrane surface, known to be approximately 20-60 nm thick (Fabrega et al., 2012, Reprod Fertil Dev 24, 619-630; Schroter et al., 1999, Hum Reprod Update 5, 302-313; and Tecle and Gagneux, 2015, Mol Reprod Dev 82, 635-650). Further experiments are under investigation to confirm this mechanism and the physiological significance behind it.

[0176] It was found that the average Ca mass of sperm treated with the DGC capacitation procedure was also significantly increased compared to control sperm diluted from the original semen conditions, whereas no change in Ca mass content was detected in sperm with abnormal oligoasthenospermia (Figure 13B). Furthermore, compared to normal sperm under control conditions, the K-average mass of oligoasthenospermia was significantly increased, whereas the K-average mass of oligoasthenospermia samples capacitated by DGC was decreased. Compared to normal controls, the fold change in the average mass of essential elements K, Mg and Zn after the DGC capacitation procedure was increased, whereas only Zn was increased in oligoasthenospermia (Figure 13D).

[0177] To verify the accuracy of this sc-ICP-MS method, 1 × 10 6 The single cell average mass results of samples with sperm densities below 100 were compared with bulk analysis by applying the conventional acidic cell digestion ICP-MS method. The cellular elemental contents determined by this sc-ICP-MS analysis and by the commonly used acidic digestion of batches of cells were within comparable ranges (Figures 14B-14C). Consistently, the average content of Ca in viable motile sperm of samples capacitated in DGC conditions was significantly increased when comparing normal sperm diluted from the original semen samples.

[0178] FIG. 15 provides the elemental mean mass profile characteristics in normal single human sperm compared to somatic cells.

[0179] To investigate whether the mean mass elemental profile was specific in normal single human sperm, the elemental profile was determined in cultured somatic cells, including WT mouse epididymal epithelial DC2 cells, embryonic human embryonic kidney 293T cells, human cervical cancer HeLa cells, and human gastric cancer SNU-1 cells (Figure 15). The mean mass results of different cell types showed that the overall content of essential elements in human sperm was generally lower compared to somatic cells, especially compared to DC2 cells, HELA cells, and HEK293T cells. The contents of Ca and Fe were statistically lower, whereas Se was substantially higher in mouse WT DC2 epithelial cells and embryonic human HEK293T cells than in human normal sperm cells. Interestingly, significantly lower amounts of Fe or Se content were observed in cancerous HeLa and / or SNU-1 cells compared to WT DC2 cells and embryonic HEK293T cells.

[0180] Figures 16-21 provide element-Ca relationships in single cells of the same biological samples.

[0181] Ca homeostasis is essential for sperm fertilization function and male fertility. Studies suggest that Ca interacts with Pb and that low Pb exposure results in low sperm motility and asthenozoospermia risk (Zhang et al., 2021, Cell Biosci 11, 150). Metal ion homeostasis, especially the transition metals Fe and Cu, play an essential role in male reproduction, but their role is a two-sided coin (Mirnamniha et al., Nelson, and Tvrda et al.). The interactions and underlying physiological relevance of elements are still largely unknown. As a first attempt to understand the interrelationship profiles between elements, a correlation analysis was performed on the relationship of the contents of the determined elements compared to Ca in single cells of sperm and somatic cells. In the correlation plots of elements against Ca in human sperm, the average contents of essential elements including Na, Zn, Co, Cr, Fe, Cu, Mn and Se were found to correlate with the content of Ca (Figures 16A-16B and 17A-17B). No correlation was observed for the essential macrometals K and Mg. Furthermore, an inverse correlation between the contents of toxic elements Ag, Cd, Ni and Pb and the content of Ca was also observed (Figures 16C and 17C). The correlation of the average mass of elements against Ca in cultures of somatic cells was also plotted (Figures 18-21). The results demonstrate that Ca homeostasis is important for Na-related nutrient homeostasis and redox-sensitive element-related physiological processes as well as toxic element detoxification. Further experiments are under investigation to confirm the physiological significance of the correlations between elements and specific elementomics at the single cell level and to explore the molecular mechanisms underlying elementomics network-related physiological processes in normal and abnormal sperm and somatic cells.

[0182] Figures 22-25 provide the association between elementomic characteristics and oligoasthenospermia risk.

[0183] The data obtained by sc-ICP-MS provided distribution patterns of the average mass of elements in individual cells, reflecting differences in cells in the same population. Based on the average mass of elements in the entire population of single sperm, no association was observed between oligoasthenospermia risk and the average mass content of elemental content in sperm. Further evaluated were the average mass frequency distribution patterns of certain elements in individual sperm prepared from normal or oligoasthenospermia semen samples, treated or not with the capacitation procedure in HTF solution with density gradient centrifugation. The results showed that almost all distribution patterns of the determined elements in individual sperm did not follow a perfect Gaussian distribution, including essential macro- and trace elements such as Na, K, Ca, Mg, Zn, Fe, Cr, Cu and Mn. For some essential trace elements such as Co and Se, as well as toxic or other elements such as Al, Cd and Ni, the frequency distribution can be described by a Poisson distribution, assuming a completely random arrival of ions to the detector, as previously described for ICP-MS spike event detection (Cornelis and Hassellov, 2014, J Anal Atom Spectrom 29, 134-144, and Wang et al., 2015). Statistical analysis showed that the distribution patterns of Na, Ca, Mg, Zn and Al in samples prepared with normal semen treated by the capacitation procedure were significantly different compared to the control group. Furthermore, the distribution patterns for K, Fe, Cr, Cu and Se in sperm with abnormal oligoasthenospermia were significantly different from those of the control group.

[0184] Taking the distribution pattern of the mean Fe mass content as an example, the results showed that in the whole defined sperm population of a single preparation of normal semen, regardless of the challenge of the capacitation procedure, it consisted of various subpopulations, where at least one with low Fe content and another with high Fe content were present (Figure 23A). In both high and low populations of signals of the same biosample, subpopulations were also preferentially identified in normal than in abnormal sperm with oligoasthenozoospermia (Figure 23B). In abnormal oligoasthenozoospermia sample preparations, no subpopulations with high Fe content were observed, regardless of the presence or absence of capacitation challenge (Figure 23A). Statistical analysis showed that the Fe distribution pattern changed significantly after the capacitation induction procedure compared to the control group of normal sperm (Figure 23A). In abnormal sperm, the distribution was statistically significant compared to the normal semen control group, but no differences were found between the capacitated abnormal sperm and the original abnormal sperm without capacitation. Interestingly, the distribution pattern of abnormal sperm after capacitation was more random than that of noncapacitated sperm. These results demonstrate that elemental distribution patterns revealed that elemental distribution patterns are a factor associated with oligoasthenozoospermia risk, especially Fe content in single sperm, consistent with the concept of metal homeostasis in regulating sperm function and male fertility (Mirnamniha et al., Nelson, and Tvrda et al.).

[0185] Unique elemental dynamics of ICP-MS spike signals were identified in normal sperm, oligoasthenozoospermia sperm, and somatic cancer cells.

[0186] To determine whether there are differences in the dynamics of certain types of elements between normal and oligoasthenozoospermic sperm (FIG. 24A), the dynamic kinetic parameters ICP-MS spike signal characteristics were further evaluated using Fe and Cu in normal sperm as examples, as shown in FIG. 5. These parameters include spike signal dwell time, which consists of the pre-peak and post-peak times to reach or shorten from the peak of a single signal within the dwell time, respectively, and the parameters of dynamic kinetic constants before and after the spike peak within the dwell time, as well as the area under the defined peak. In an attempt to define the characteristics, the exported ICP-MS spike signal traces were analyzed and the parameters were determined manually. For the kinetics before and after the spike peak with a single signal, the time constants were obtained by fitting the rise or shortening of the pre- or post-peak kinetics with a standard alpha exponential function.

[0187] The peak style patterns of sc-ICP-MS elemental signals were evaluated using Fe, Zn, and Cu as examples, where Fe represents an example of an element with an asymmetric tailing peak style, and Cu represents an example of an element with a peripheral symmetric peak style (Figure 24A). The results showed that the post-peak residence time of Fe in sperm was significantly increased after challenge with the capacitation procedure, regardless of whether they were normal or abnormal oligoasthenozoospermia sperm. No difference in residence time was observed between normal and abnormal sperm. Comparing normal control sperm with capacitation-stimulated sperm, the post-peak tau constant of abnormal sperm was substantially decreased. For Cu, the residence time was shortened in abnormal sperm compared to the normal group under non-capacitation conditions, but there was no difference in residence time or tau constant between the two groups after capacitation stimulation. For Zn, compared to normal or non-capacitated sperm, the total spike residence time of normal sperm was significantly higher than that of the abnormal group, either before or after capacitation stimulation. Regarding Zn tau constant, it was significantly decreased after capacitation in the abnormal group compared with normal sperm.

[0188] The kinetic dynamics of certain kinds of elements in normal human sperm, including Cr, Fe, Zn, Cu, Mn and Se, were also summarized for comparison (Figure 24B). The residence times for Cr and Fe were significantly higher than other elements such as Zn, Cu, Mn and Se, and Zn was also significantly higher than Cu, Mn and Se. The tailing tau constants of Cr and Fe were significantly higher than other elements. The physiological implications of this special kinetic profile in sperm are still under investigation.

[0189] To determine whether the single element ICP-MS signal characteristics are specific to sperm, the residence times (full peak, pre-peak and post-peak residence times) and dynamic peak-related kinetic constants of essential trace elements in other cultured somatic cells were further evaluated (Figure 25). In general, the residence times for Fe and Cr elements were substantially longer than those of Zn, Cu and Mn elements, regardless of cell type. Compared to normal human sperm, the full peak and post-peak residence times of Zn, Fe, Cu and Cr were significantly different from those of various somatic cells, but the pre-peak residence times were not. No difference was observed in the residence time of Mn between human sperm and various somatic cells. With regard to the time constants, the post-peak kinetic tau constants for Fe, Cu and Cr elements in human sperm were significantly different from those of other somatic cells. Regarding pre-peak residence times, only Mn element was significantly different in SNU-1 cells compared to human sperm and 293T cells, whereas no differences were observed for Zn, Fe, Cu and Cr elements in the tested cell types.

[0190] Overall, these results showed for the first time that different dynamic ICP-MS peak patterns and kinetics of different distributions of different elements in several somatic cells, including sperm cells and cancer cells, are distinct and potentially related to pathological conditions. In support of this hypothesis, the results showed that single ICP-MS spike signal characteristics of elemental elements in single sperm correlate with the risk of oligoasthenospermia. Without wishing to be bound by theory, different peak patterns of different elements in the same cell population represent unique elemental features of a given specific cell type that may be related to their status under defined physiological or pathophysiological conditions. The signal values ​​and kinetic characteristics of these elements have biotoxicological and pathological significance and may be used in clinical diagnosis.

[0191] The same parameters of other investigated elements of all cell types examined in this study are also determined using the same analytical approach. These parameters include all the characteristics of a specific elemental signal of a given cell type with its unique elemental and morphological characteristics. Experiments are under investigation to confirm the clinical significance and other potential applications of these characteristic parameters, such as diagnosis or prognosis of male infertility or cancer or other diseases.

[0192] Additional experiments are under investigation to confirm the physiological and pathological significance of elemental subpopulations in single human sperm cells under physiological or pathological conditions for clinical diagnosis.

[0193] To obtain sensitivity and accurate signal of single sperm cells by ICP-MS, the sperm number density is controlled and only one cell is transported to plasma at any given time, which is determined by the residence time. In Figures 34A-34I, it is shown that the signal profile of elemental Fe increases as the cell density loaded into the ICP-MS for measurement increases. The cell density is determined by the number of cell events exceeding 3 x 10 per mL. 4 ~About 2×10 per mL 6While the cellular events increased linearly in the range of 0.01 to 0.1 × 10 per mL, reflecting overlapping cellular signals, ... although not wishing to be bound by theory. 6 To avoid false positive signals, the cell density was reduced above 1 × 10 per mL. 6 were excluded from statistical analysis. No obvious changes in cellular events were observed over 20 h after fixation of sperm cells and samples were stored in pure water before measurements.

[0194] Example 3: Analysis of dynamic elemental profiles during capacitation of mouse sperm using single-cell ICP-MS Analysis of metal ion flux during capacitation of mouse sperm using single-cell ICP-MS:Currently, clinical analysis of male infertility mainly relies on semen analysis and sperm parameters. However, high diagnostic failure rates indicate that current evaluation methods remain insufficient and new approaches to assess sperm function still need to be developed. With increasing evidence showing the role of biometals in reproductive biology, the importance of metal ion homeostasis in sperm function and male fertility is emerging. To determine the changes in metals in single sperm cells during fertilization activity, single-cell inductively coupled plasma mass spectrometry (sc-ICP-MS) technology was utilized to measure metal concentrations in capacitated sperm. In this study, we used a male sterile calcium pump PMCA4 knockout mouse model with calcium dysregulation during fertilization events. Consistently, the results showed abnormal dynamic calcium profiles in PMCA4-KO sperm undergoing capacitation. Overall, this study demonstrates that sc-ICP-MS can be applied to sperm function analysis.

[0195] Materials and Methods Materials and Reagents: All reagents used were analytical grade or of the highest quality available. Sodium chloride, potassium chloride, and potassium dihydrogen phosphate were purchased from Sinopharm Company (Shanghai, China). Nitric acid (65%), calcium chloride, magnesium sulfate heptahydrate, glucose, HEPES, sodium lactate, sodium pyruvate, sodium bicarbonate, and phenol red were all purchased from Sigma-Aldrich Company (St. Louis, MO USA), and BSA was purchased from BBI Life Science Company (Shanghai, China). Single-cell ICP-MS spherical 60 nm gold nanoparticle elemental standards were supplied by the sc-ICP-MS analyzer manufacturer (PerlinElemer, Shanghai, China). Ultrapure water (18.2 MΩ) was used. Standard solutions of desired concentrations were prepared daily using serial dilutions of stock solutions.

[0196] Animals: Pmca4 knockout (KO) mouse models were obtained from the Delaware Biotechnology Institute, University of Delaware, USA, and housed at the Shanghai Research Center for Model Organisms. All animal experiments were performed in accordance with the guidelines for the use of laboratory animals established by the Animal Ethics Committee of ShanghaiTech University. Male Pmca4 - / - These mice were generated using heterozygous pairing because they were germ-free. In this study, 8- to 12-week-old Pmca4 mice were + / + (wild type, WT) and Pmca4 - / -(Knockout, KO) C57BL / 6 mice were used. The genotype of Pmca4 KO mice was identified using PCR using the forward primer (5'-CTGTGGGAACCCCGTTGGTCTCTTTC-3') and reverse primer (5'-GCACCCAGGCGATGGATGGCAAAGCT-3') as previously reported (see, e.g., Okunade et al., 2004, J Biol Chem 279, 33742-33750).

[0197] Sperm capacitation medium: HEPES-buffered medium was used throughout the study for the preparation and capacitation of Pmca4 KO mouse sperm. The composition of the HEPES-buffered medium was 95 mM NaCl; 5 mM KCl; 1.7 mM CaCl. 2 ;1.2 mM MgSO 4 7H 2 HO; 1.2 mM KH 2 PO 4 20 mM sodium lactate (60%); 0.27 mM sodium pyruvate; 25 mM NaHCO 3 50 mM glucose; 3 mg / mL BSA; 20 mM HEPES and 0.02 mg / mL phenol red. All solutions were made with HEPES, BSA, NaHCO 3 or CaCl 2 The HEPES buffer was sterilized by passing it through a 0.22 μm filter and warmed on a metal block at 37° C. prior to use.

[0198] Preparation of sperm samples: Mice were sacrificed by anesthesia with sodium pentobarbital, and the epididymis was rapidly removed. Sperm were then obtained for capacitation assays as previously reported (see, e.g., Zi et al., PLoS Genet, 2015, 11, e1005485, and Ma et al., iScience, 2019, 14, 210-225). Briefly, the caudal region of the epididymis was cut twice before being gently shaken in HEPES-buffered capacitation medium, which allowed sperm to flow out of the epididymal tubules, and the epididymis was removed after shaking. The HEPES-buffered capacitation medium containing sperm was placed in a metal bath at 37 °C for 5 min, and the time was assigned as 0 h. At the indicated time points, sperm were immediately aspirated with a 1 ml injection pump (Harvard Apparatus) into a suction chamber for single-cell ICP-MS analysis.

[0199] Instrumentation: For single cell ICP-MS experiments, a quadrupole-based Perkin-Elmer NexION 2000 ICP-MS equipped with a single cell analysis module and an Asperon™ single cell spray chamber was used under the operating conditions listed in Table 4. Prior to analysis, capacitated sperm were first collected at the indicated time points by centrifugation to remove the capacitation buffer. Sperm were resuspended by adding an appropriate volume of HEPES-buffered capacitation solution and separated using a hemocytometer for 10 min before loading for ICP-MS analysis. 5 The suction chamber was washed between samples using 3% nitric acid followed by deionized water.

[0200] [Table 4]

[0201] Statistics: All experiments were repeated at least three times using independent WT and Pmca4 KO mouse sperm samples. Data are presented as mean ± SD. Two-way ANOVA was performed to compare differences between multiple comparisons. P values ​​of 0.05 or less were considered significant. Skewness and kurtosis parameters of frequency distributions for sperm mass populations were calculated using Prism software (GraphPad).

[0202] Results and Discussion Optimization of experimental conditions Regarding sc-ICP-MS analysis, it is important to optimize the conditions under which the cells maintain their intact cellular morphology and remain monodispersed, so that each ICP-MS spike corresponds to a single cell event. Optical microscopy showed that the sperm cells maintained their morphological integrity and were monodispersed after aspirating into the spray chamber before loading into the ICP-MS plasma chamber for elemental detection (Figure 26). As the sperm were immersed in sodium-rich capacitation medium, the ratio of its extracellular to intracellular concentration was too high for single cell analysis, and the interference of potassium ions was severe without other scavenging gases in the reaction chamber, the content of these two ions was then excluded from the analysis in this study.

[0203] Calcium spectroscopy shows calcium overload in single Pmca4 KO spermatids In sperm, the capacitation process is initiated by the influx of intracellular Ca 2+ Postcapacitation homeostasis in the cytoplasm is maintained by calcium efflux via the Pmca4 pump.

[0204] Thus, Pmca4 deficiency reduces resting intracellular Ca 2+This leads to an increase in calcium concentration and consequent impairment of sperm motility (see, for example, Navarrete et al., J Cell Physiol, 2015, 230, 1758-1769, and Schuh et al., 2004, J Biol Chem 279, 28220-28226). The results showed that the average resting calcium content in single Pmca4 KO sperm cells was slightly higher than that in WT cells. Before capacitation stimulation, the average content at time 0 was found to be 2881 ± 256 attograms in single WT sperm and 3225 ± 221 attograms in Pmca4 KO sperm, slightly decreased to 2556 ± 212 attograms in WT sperm, but increased to 3903 ± 1375 attograms in Pmca4 KO sperm after 2 hours in capacitation physiological conditions (Figure 27A). Overall calcium content in Pmca4 KO sperm was significantly and progressively increased compared to WT controls (P<0.05), especially after 2 h of capacitation, although the increase was not significant at earlier time points (Figure 27A). As capacitation progressed toward 2 h, the frequency distribution of calcium mass in single cells was altered in Pmca4 KO sperm compared to control WT sperm (Figures 27B-27F). The results showed that the data distribution at time 0 essentially showed a normal distribution for frequency distribution measurements of calcium content in single Pmca4 KO sperm cells compared to WT controls. Thus, the increase in calcium content in Pmca4 KO sperm cells was due to the increased calcium mass in single Pmca4 KO sperm cells compared to WT controls. 2+ The ions are not pushed out, and some sperm cells have more Ca than others. 2+ This is consistent with the notion that Pmca4 accumulates Ca2+ in the sperm cells. Interestingly, skewness and kurtosis parameters were significantly increased at 2 h of capacitation (P<0.05) (Figures 27G-27H). Skewness and kurtosis are measures of the symmetry of the frequency distribution and the degree of tailedness therein, respectively. In other words, skewness measures the relative size of the two tails, whereas kurtosis measures the combined size of the two tails of the frequency distribution. Thus, the significant positive changes in skewness and kurtosis in Pmca4 KO sperm cells are consistent with the notion that Pmca4 accumulates Ca2+ in the sperm cells via the Pmca4 pump.2+ Consistent with a failure of ion extrusion, some sperm cells had more Ca than others. 2+ As analyzed by sc-ICP-MS in the same single sperm populations of Pmca4 KO and WT mice, the range of Ca average mass content (in attograms) in single live sperm cells changed dynamically at different time points under capacitation conditions, especially at the initial zero time point and 1.5 or 2 h under capacitation conditions (Figure 27I). These results suggest that the Ca content in capacitated sperm cells in Pmca4 KO sperm is significantly increased. 2+ This is consistent with the observation of increased calcium content in sperm, demonstrating that the single-cell ICP-MS technique is capable of determining sperm calcium content under physiological and pathological conditions.

[0205] Altered elemental dynamics in single sperm cells of Pmca4 KO mice during the capacitation process Analysis of the mean mass and frequency distribution of the mean mass of elements Zn, Fe, Cu, Mn, and Se in single sperm of Pmca4 KO sperm cells showed no obvious differences compared to WT sperm during capacitation (Figures 28A-28F, 29A-29F, 30A-30F, 31A-31F, 32A-32F), but the range of the mean mass content of these elements showed obvious altered dynamics in single live sperm cells of Pmca4 KO mice compared to WT sperm under capacitation conditions, and the dynamic changes were particularly prominent for Fe, moderate for Zn, and mild for Cu, Mn, and Se elements (Figures 28I, 29I, 30I, 31I, and 32I).

[0206] Altered skewness and kurtosis parameters of dynamic frequency patterns of zinc and manganese ions in single Pmca4 KO sperm cells Exogenous zinc (Zn) has been reported to cause disruption of calcium homeostasis and cell death, as well as increased ROS levels (see, e.g., Guo et al., Arch Biochem Biophys, 2014, 560, 44-51). In addition, high levels of manganese have been reported to have a detrimental effect on human sperm viability and motility (see, e.g., Li et al., 2012b), while trace amounts of manganese have been reported to provide antioxidant protection for sperm cryopreservation (see, e.g., Cheema et al., Oxid Med Cell Longev, 2009, 2, 152-159). In this study, although the measured masses of Zn or manganese (Mn) in single sperm cells of Pmca4 KO mice were not significant compared to WT mice, the skewness and kurtosis parameters for the frequency distribution of Zn and Mn ions showed significant variations (P<0.05) during the process of capacitation (Figures 28 and 31).

[0207] These results suggest that accumulated intracellular Ca 2+ We demonstrate that Zn- or Mn-related redox states in Pmca4 KO sperm may be dynamically altered, and therefore experiments are under investigation to determine whether this correlates with certain cases of clinical idiopathic male infertility.

[0208] Distinct dynamic patterns of metallomics in single sperm cells In order to elaborate the elementomic characteristics of Pmca4 KO sperm cells, in addition to measuring and analyzing the average mass and dynamic patterns of the macro and rare trace elements in the same sperm samples, including calcium (Figure 27), zinc (Figure 28), iron (Figure 29), copper (Figure 30), manganese (Figure 31), and selenium (Figure 32) ions under the sperm capacitation process, in an attempt to further elaborate the elementomic characteristics of specific elements in single sperm cells, the ratio of the molar content of each element in single sperm cells to the nickel element of each sample was determined and demonstrated in Figures 33A-33F. We used the average molar mass of nickel element as the allocation because its content is relatively low and stable and does not change during the capacitation process. The results showed that the molar ratio of calcium concentration to nickel concentration in single sperm was significantly different between the Pmca4 KO group and the WT group 2 hours after capacitation (P<0.05). These results demonstrate that ratiometric analysis of elemental contents of the same sample can further reveal the importance of elemental properties in single cells.

[0209] conclusion The present study represents the first step towards the application of sc-ICP-MS in the elementomic analysis of single sperm cells. This study demonstrates the feasibility of sc-ICP-MS becoming a commonly used analytical tool for evaluating calcium and other essential metal content, as well as dynamic elemental patterns, of single sperm under physiological processes such as sperm capacitation, thereby assessing sperm functional quality and ultimately individual male fertility. The method is rapid, simultaneously characterizes various elements, and involves a relatively simple procedure. However, maintaining the accuracy of sperm integrity before the experimental procedure and after the experimental algorithmic analysis remains the focus and challenge of the clinical application of this technique. Nevertheless, the study herein shows the potential application of sc-ICP-MS to the clinic for the evaluation of human sperm functional quality, providing a multifaceted reference for clinical diagnosis.

[0210] Although certain embodiments have been illustrated and described, those skilled in the art, after reading the foregoing specification, may effect modifications, substitutions of equivalents, and other types of changes to the methods of the technology described herein. Each of the above aspects and embodiments may also include or incorporate variations or features as disclosed with respect to any or all of the other aspects and embodiments.

[0211] The present technology is not limited with respect to the specific embodiments described herein, which are intended as single illustrations of individual embodiments of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, labeled compounds or biological systems, which, of course, may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting. Thus, it is intended that the specification be considered exemplary only, with the breadth, scope, and spirit of the present technology being indicated only by the appended claims, the definitions therein, and any equivalents thereof.

[0212] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," and the like, should be read broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description, not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. In addition, the phrase "consisting essentially of" will be understood to include the elements specifically recited, as well as additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of" excludes any elements not specified.

[0213] In addition, where features or aspects of the disclosure are described in terms of Markush groups, one of skill in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group. Each of the narrower species and subgeneric groupings included in the generic disclosure also form part of the invention. This includes the generic specification of the invention with a provisos or negative limitation removing any subject matter from that genus, regardless of whether the removed material is specifically described herein.

[0214] As will be understood by those skilled in the art, for any or all purposes, especially in terms of providing a written description, all ranges disclosed herein also include any and all possible subranges and combinations of those subranges. Any recited range can be easily recognized as fully describing and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily broken down into a lower third, a middle third, an upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," etc. refers to a range that includes the recited numbers and can then be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member.

[0215] All publications, patent applications, issued patents, and other documents (e.g., journals, articles, and / or textbooks) mentioned herein are hereby incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the texts incorporated by reference are excluded to the extent they conflict with definitions in the present disclosure.

[0216] It is to be understood that the present technology may include, but is not limited to, the features and combinations of features recited in the following paragraphs, and that the following paragraphs should not be construed as limiting the scope of the claims appended hereto or as mandating that such features necessarily must be included within the scope of such claims. A. A method for detecting multiple trace elements in a trace cell, comprising the steps of: Step 1: Obtain somatic cells, centrifuge the cells, remove the supernatant, and solubilize the centrifuged cells in diluent to 3 × 10 6The cells are suspended to a detection concentration of less than 1 / ml and the suspended cells are loaded into an sc-ICP-MS instrument for detection. Step 2: The detection method is carried out using conventional sc-ICP-MS techniques. A single-cell nebulizer is used to aspirate the cell suspension into droplets in a single-cell spray chamber, each of which constitutes a single cell, and a rapid analysis digitization software module is used to analyze multiple trace elements at the single-cell level. Step 3: Using the same procedure in step 2, detect a series of standard solutions with known concentrations of elements and plot a standard curve. Step 4: Determine the unique elemental ICP-MS signal characteristics of a particular cell type using the method, which includes evaluating the sc-ICP-MS signal kinetics characteristics of a particular element in a particular cell selected from dwell time, peak time of a single signal spike, ratio of pre-peak or post-peak time of the spike relative to dwell time, dynamic kinetic constant or pre-peak area of ​​the spike, dynamic kinetic constant or post-peak area of ​​the spike, etc. Step 5: Assess the functional quality of the cells. B. A method for assessing the functional quality of a cell, characterized by assessing the dynamic kinetic properties of sc-ICP-MS signal spikes of a specific element in a specific cell, selected from dwell time, peak time of a single signal spike, ratio of peak time to dwell time, dynamic kinetic constant of the spike or area before the peak, or dynamic kinetic constant of the spike or area after the peak. C. Use of the method for detecting multiple trace elements in trace cells according to item A in the evaluation of human sperm cells. D. Use of the method for detecting multiple trace elements in trace cells described in item A in evaluating various forms and types of cells selected from whole somatic cells, gamete cells, prokaryotic cells of any genus, or isolatable cells in semen, reproductive tract fluid, follicular fluid, blood, urine, saliva or feces. E. Use of the method for assessing the functional quality of cells according to item B in assessing human sperm cells. F. Use of the method for assessing the functional quality of cells according to item B in assessing various morphologies and types of cells selected from total somatic cells, gamete cells, prokaryotic cells, or isolatable cells in semen, reproductive tract fluid, follicular fluid, blood, urine, saliva or feces. G. A method for detecting multiple trace elements in human sperm cells, comprising the steps of: Step 1: Sample preparation: Liquefy fresh human semen according to conventional semen preparation methods, centrifuge the liquefied semen to separate seminal plasma and sperm, resuspend the sperm in 4% PFA, fix the sperm in 4% PFA for 15 minutes, remove the fixative by washing, and perform the following steps with the sperm or store the sperm at 4°C for later use. Step 2: Dilute the sperm to 3 x 10 6 Dilute to a detection concentration of less than 1 / ml. Step 3: Load the sample into the sc-ICP-MS instrument for detection. A specific single-cell nebulizer and spray chamber were installed and calibrated with an adjustment solution (2% v / v nitric acid containing 10 μg / L Li, Be, Mg, Fe, In, Ce, Pb and U) to determine the appropriate instrument detection parameters; gold particle standards were used to determine the single-cell transmission efficiency, which is generally in the range of 40%-60%; standard solutions with different concentrations of different elements were prepared and standard curves were plotted; all standard solutions were prepared in ultrapure water; to eliminate polyatomic interferences and obtain a high signal-to-noise ratio, measurements were performed in dynamic reaction cell (DRC) mode using ammonia as a reactant gas for the detection of K, Ca, Cr, and Fe in single cells, or using oxygen as a reactant gas for the detection of As and Se in single cells by detecting the oxidation reaction products of AsO and SeO as analytes; other elements were measured in standard mode; the sample spike signal was sampled for 50 s with a dwell time of 50 μs (i.e., digitization frequency at 50 MHz); the total sample volume consumed by detecting 18 elements was approximately 400 μL. Step 4: Determine the unique elemental ICP-MS signal characteristics of a particular cell type using the method, which includes evaluating sc-ICP-MS signal kinetics characteristics of a particular element in a particular cell selected from dwell time, peak time of a single signal spike, ratio of pre-peak or post-peak time of the spike relative to dwell time, dynamic kinetic constant or pre-peak area of ​​the spike, dynamic kinetic constant or post-peak area of ​​the spike. Step 5: Assess the functional quality of the cells

[0217] Other aspects are within the scope of the following claims.

Claims

1. 1. A method for detecting infertile sperm in a sample obtained from a subject, the method comprising detecting a concentration of at least one metal in the sample that is outside a predetermined range using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS).

2. The method of claim 1, wherein the at least one metal is selected from the group consisting of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni).

3. The method described in claim 1 or 2, wherein the sample is diluted with a buffer solution before the detection step.

4. The method of claim 1, wherein the predetermined range corresponds to the metal concentration detected in sperm from a population of fertile subjects.

5. The method described in claim 1, wherein the sperm have acquired fertilization capacity.

6. The method described in claim 1, wherein the sperm have not acquired fertilization capacity.

7. The predetermined range of Na concentration is from about 5 attograms (ag) to about 50,000 ag for non-capacitated sperm, and from about 25 ag to about 50,000 ag for capacitated sperm; the predetermined range of K concentration is from about 50 ag to about 50,000 ag for uncapacitated sperm and from about 280 ag to about 50,000 ag for capacitated sperm; the predetermined range of Ca concentration is about 200 ag to about 50,000 ag for uncapacitated sperm and about 700 ag to about 20,500 ag for capacitated sperm; the predetermined range of Mg concentration is from about 8 ag to about 50,000 ag for uncapacitated sperm and from about 75 ag to about 15,100 ag for capacitated sperm; the predetermined range of Zn concentration is from about 5 ag to about 50,000 ag for uncapacitated sperm and from about 20 ag to about 50,000 ag for capacitated sperm; the predetermined range of Fe concentration is from about 5 ag to about 50,000 ag for uncapacitated sperm and from about 13 ag to about 50,000 ag for capacitated sperm; the predetermined range of Al concentration is from about 3 ag to about 50,000 ag for uncapacitated sperm and from about 6 ag to about 46,700 ag for capacitated sperm; the predetermined range of Se concentration is from about 59 ag to about 50,000 ag for uncapacitated sperm and from about 62 ag to about 45,810 ag for capacitated sperm; the predetermined range of Co concentration is from about 3 ag to about 3,700 ag for uncapacitated sperm and from about 9 ag to about 20,200 ag for capacitated sperm; the predetermined range of Cu concentration is from about 9 ng to about 50,000 ag for uncapacitated sperm and from about 9 ng to about 37,590 ag for capacitated sperm; the predetermined range of Cr concentration is from about 4 ng to about 50,000 ag for uncapacitated sperm and from about 5 ag to about 46,700 ag for capacitated sperm; 2. The method of claim 1, wherein the predetermined range of Mn concentration is from about 2 ag to about 50,000 ag for uncapacitated sperm and from about 7 ag to about 32,610 ag for capacitated sperm.

8. A method for detecting infertile sperm in a sample obtained from a subject, the method comprising detecting dynamic or kinetic parameters of signal spikes of at least one metal selected from the group consisting of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni) in the sample that are outside a predetermined range by single cell inductively coupled plasma mass spectrometry (sc-ICP-MS).

9. The method of claim 8, wherein the predetermined range corresponds to a dynamic or kinetic parameter detected in sperm from a population of fertile subjects.

10. The method described in claim 8 or 9, wherein the dynamic or kinetic parameter of the spike is selected from dwell time, pre-peak dwell time, post-peak dwell time, peak time, the ratio between the peak time and the dwell time, the rising tau constant before the peak, the dynamic area before the peak, the tailing tau constant after the peak, the dynamic area after the peak, or any combination thereof.

11. The dynamic or kinetic parameters of the spikes: (a) for uncapacitated sperm, a dwell time of the Fe spike of about 1.4 to about 7.9 ms, and for capacitated sperm, a dwell time of the Fe spike of about 1.5 to about 6.7 ms; (b) for uncapacitated sperm, a tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.81 ms, and for capacitated sperm, a tailing tau constant after the peak of the Fe spike of about 0.18 to about 0.90 ms; (c) for uncapacitated sperm, an elevated tau constant before the peak of the Fe spike of about −0.35 ms or less, and for capacitated sperm, an elevated tau constant before the peak of the Fe spike of about −0.80 ms; (d) a Cu spike dwell time of about 1.5 ms or less for uncapacitated and capacitated sperm; (e) for uncapacitated sperm, an elevated tau constant before the peak of the Cu spike of about −0.2 ms or less, and for capacitated sperm, an elevated tau constant before the peak of the Cu spike of about −0.6 ms or less; (f) a tailing tau constant after the peak of the Cu spike of about 0.15 ms or less for uncapacitated sperm, and a tailing tau constant after the peak of the Cu spike of about 0.2 ms or less for capacitated sperm; (g) for uncapacitated sperm, a dwell time of the Zn spike of about 2.1 ms or less, and for capacitated sperm, a dwell time of the Zn spike of about 1.2 ms or less; (h) for uncapacitated sperm, an elevated tau constant before the peak of the Zn spike not greater than about −0.25 ms, and for capacitated sperm, an elevated tau constant before the peak of the Zn spike not greater than about −0.20 ms; (i) for uncapacitated sperm, a tailing tau constant after the peak of the Zn spike of about 1.15 ms or less, and for capacitated sperm, a tailing tau constant after the peak of the Zn spike of about 0.25 ms or less; (j) a dwell time of the Cr spike of about 3.25 ms or less for uncapacitated sperm, and an elevated tau constant before the peak of the Cr spike of about −0.45 ms or less for uncapacitated sperm; (k) for uncapacitated sperm, a tailing tau constant after the peak of the Cr spike from about 0.2 to about 0.5 ms; (l) for uncapacitated sperm, a residence time of the Se spike of about 1.5 ms or less; (m) for uncapacitated sperm, an elevated tau constant before the peak of the Se spike of about −0.15 ms or less; and 11. The method of claim 10, comprising: (n) for uncapacitated sperm, a tailing tau constant after the peak of the Se spike of about 0.25 ms or less.

12. A method for detecting infertile sperm in a sample, comprising: (i) contacting said first population of sperm from a subject with HTF (human tubal fluid) buffer; and (ii) using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) to detect a concentration of at least one metal selected from the group of potassium (K), calcium (Ca), magnesium (Mg), mercury (Hg), silver (Ag), and aluminum (Al) in said first population of sperm after the contacting step that is equal to or lower than the concentration present in a second population of sperm that have not been contacted with HTF buffer; or (iii) using single cell inductively coupled plasma mass spectrometry (sc-ICP-MS) to detect a concentration of at least one metal selected from the group of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), selenium (Se), cobalt (Co), chromium (Cr), cadmium (Cd), manganese (Mn), arsenic (As), mercury (Hg), lead (Pb), silver (Ag), aluminum (Al), and nickel (Ni) in said first population of sperm after the contacting step that is equal to or lower than the concentration present in a second population of sperm that have not been contacted with HTF buffer; or (iv) using the sc-ICP-MS to detect a concentration of selenium (Se) in the first population of sperm after the contacting step that is equal to or greater than the concentration present in the second population of sperm that have not been contacted with HTF buffer; or (v) A method comprising both (ii), (iii), and (iv).

13. A method described in any one of claims 1, 8 or 12, wherein the sample is obtained from a subject.

14. The method of claim 13, wherein the subject has or is suspected of having idiopathic infertility, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia.

15. The method of claim 13, further comprising treating the subject with an infertility therapy or procedure.

16. The method described in claim 15, wherein the concentration of at least one metal in the sperm is lower than the predetermined range and the infertility therapy includes treatment with the at least one metal.

17. The method of claim 1, 8 or 12, wherein two or more of the metals are detected.

18. The method of claim 1, 8 or 12, wherein the sperm are diluted to a concentration of 3 x 10 6 sperm / ml or less prior to the detecting step.

19. A method described in any one of claims 1, 8 or 12, wherein the sperm are diluted 10 times or more prior to the detecting step.

20. The method of claim 18, wherein the sperm are centrifuged to remove seminal plasma before dilution.

21. The sample, optionally, Liquefied or Fixed or Capacitated or cryopreserved or Liquefied and fixed, or Liquefied and capacitated, or Liquefied and cryopreserved, or fixed and capacitated, or Fixed and cryopreserved, or Capacitated and cryopreserved, or Liquefied, fixed and capacitated, or Liquefied, fixed, and cryopreserved, or Liquefied, capacitated, and cryopreserved, or Liquefied, fixed, capacitated and cryopreserved 13. The method of any one of claims 1, 8 or 12, comprising semen.

22. The method of claim 1, 8, or 12, further comprising performing computer-aided sperm analysis (CASA) on the sperm.

23. A method described in any one of claims 1, 8 or 12, further comprising purifying the sperm having (i) at least one metal concentration, or (ii) the dynamic or kinetic parameter, or both (i) and (ii), within the predetermined range to obtain functional sperm.

24. The method of claim 23, optionally further comprising fertilizing the purified functional sperm with an egg via in vivo fertilization (IVF) or intracellular sperm injection (ICSI).

25. A kit comprising a buffer solution and instructions for carrying out the method of any one of claims 1, 8 or 12.

Images