Methods for analyzing somatic cell-derived DNA molecules in a semen sample

Abstract

Methods for analyzing somatic cell-derived DNA molecules in a semen sample. The methods may involve, individually or in combination, isolating and analyzing select populations of DNA obtained from sperm cells and somatic cells in a semen sample and size-based sorting. The methods further relate to quantitative analysis of somatic cell-derived DNA molecules in a semen sample. The methods described herein also relate to diagnostic methods for detecting prostate cancer, benign prostate hyperplasia (BPH), prostatitis, or testicular cancer in isolated populations of somatic cell-derived DNA obtained from a patient.

Description

Attorney Ref.60974.00008WO01 (FLW-0006-WO) METHODS FOR ANALYZING SOMATIC CELL-DERIVED DNA MOLECULES IN A SEMEN SAMPLE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 647,994, filed May 15, 2024, entitled “Methods For Analyzing Somatic Cell-Derived DNA Molecules In A Semen Sample,” and U.S. Provisional Patent Application No. 63 / 696,212, filed September 18, 2024, entitled “Methods For Analyzing Somatic Cell-Derived DNA Molecules In A Semen Sample,” each of which are entirely incorporated herein by reference for all purposes. FIELD OF THE INVENTION

[0002] This disclosure relates to methods for analyzing somatic cell-derived DNA molecules in a semen sample. The method may involve, individually or in combination, isolating and analyzing select populations of DNA obtained from sperm cells and somatic cells in a semen sample and size-based sorting. This disclosure further relates to quantitative analysis of somatic cell-derived DNA molecules in a semen sample. This disclosure also relates to diagnostic methods for detecting prostate cancer, benign prostate hyperplasia (BPH), prostatitis, or testicular cancer in isolated populations of somatic cell-derived DNA obtained from a patient. BACKGROUND OF THE INVENTION

[0003] Prostate cancer is one of the most common cancers in the United States, and a leading cause of cancer death in men. Currently, screening for prostate disorders is conducted using blood samples and / or biopsies. Blood samples may be used for testing of Prostate Specific Antigen (PSA) levels in the blood as a biomarker for the detection and monitoring of various disease states. However, issues with this biomarker are known (e.g., over-diagnosis), limiting its accuracy and use in cancer screenings. More recently, assessment of circulating cell free nucleic acids through liquid biopsies is increasingly being used to screen for cancers. Blood samples are a common sample type used for such analysis, however in the prostate cancer context, attempts to detect prostate cancer using circulating tumor DNA in blood have not shown promising results (see, e.g., Lui et al., Ann Oncol.2020 Jun;31(6):745-759)). Further, use of liquid biopsies is subject to certain limitations, such as not having sufficient signal and thus introducing a lack of differentiation between a true negative and a false negative. Biopsies are invasive procedures that are cost intensive and / or require recovery time.

[0004] In view of the foregoing, there remains a critical need in the art to provide a reliable method of isolating DNA of interest (e.g., isolation of DNA from the prostate or testes in a background of DNA from sperm), as well as a way to improve the reliability of non-invasive diagnostic methods including prostate disorder diagnostics.Attorney Ref.60974.00008WO01 (FLW-0006-WO) SUMMARY OF THE INVENTION

[0005] Aspects of the present disclosure include methods for enriching a semen sample for a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of nucleosomal exDNA molecules derived from sperm cells.

[0006] Aspects of the invention include methods for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules from the somatic cells; centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

[0007] Aspects of the invention include methods for enriching a semen sample for a plurality of exDNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of exDNA molecules derived from somatic cells and a plurality of nucleosomal exDNA molecules derived from sperm cells, and isolating the DNA fraction, thereby enriching the semen sample for the plurality of exDNA molecules derived from somatic cells.

[0008] Aspects of the invention include methods for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules from the somatic cells and the somatic cell lysis reagent; centrifuging the mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

[0009] Aspects of the invention include methods for analyzing a quantity of somatic cell derived DNA molecules from a specific somatic cell type in a semen sample, the method comprising: isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the DNA fraction to determine an abundance of somatic cell derived DNA molecules from the specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a sufficient quantity of the somatic cell derived DNA molecules from the specific somatic cell type for analysis of a prostate-specific disorder.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0010] Aspects of the invention include diagnostic methods for detecting a prostate-specific disorder from a semen sample, the method comprising: isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the plurality of somatic cell derived DNA molecules to determine an abundance of somatic cell derived DNA molecules from a specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules derived from cells of the specific somatic cell type to a threshold value to determine whether the semen sample contains a clinically relevant prostate-specific disorder signal.

[0011] These and further aspects will be further explained in the rest of the disclosure, including the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a graph showing proportions of small molecular weight DNA as a function of the tissue from which the cell signal was received in a cohort of assumed healthy controls.

[0013] FIG.2 is a graph showing proportions of high molecular weight DNA as a function of the tissue from which the cell signal was received in a cohort of assumed healthy controls.

[0014] FIG. 3 is a graph showing the proportion of high molecular weight DNA signal as a function of the tissue from which the cell signal was received in the “run-in” for a clinical cohort.

[0015] FIG.4 is a graph showing the difference between signals received from high molecular weight DNA and small size selected DNA in the “run-in” for a clinical cohort and healthy controls.

[0016] FIGS. 5A-5B are graphs showing that sperm DNA causing background noise can be removed from analyses by performing high-speed spins, using data derived from vasectomy and non-vasectomy patients.

[0017] FIG.6 are plots derived from vasectomy and non-vasectomy patients, showing that sperm DNA causing background noise can be removed from analyses by performing high-speed spins.

[0018] FIG. 7 are plots showing that a single higher-speed spin can be sufficient for removing background noise from analyses.

[0019] FIG. 8 is a plot with tissue deconvolution data for the full clinical cohort showing that sperm signal in non-vasectomy subjects can be reduced by enriching for high molecular weight cfDNA.

[0020] FIG.9 is a table summarizing the data shown in FIG.8.

[0021] FIGS.10A-10B summarize an exemplary method for sperm sample analysis.

[0022] FIG. 11 summarizes the results of analyses performed using samples from a clinical cohort (PRISM study) as compared to assumed healthy controls (FRISKE study).

[0023] FIG. 12 summarizes the results of tissue deconvolution analysis of size selected small cfDNA by prostate cancer grade group.

[0024] FIG. 13 summarizes the results of tissue deconvolution analysis of high molecular weight cfDNA by prostate cancer grade group.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0025] FIG.14 depicts electropherogram images illustrating the stabilization of sperm and cfDNA with preservation solution.

[0026] FIG. 15 depicts cfDNA yields correlated weakly with volume. A: Total yield (ng) by volume of seminal plasma (SP) into extraction.

[0027] FIG. 16 depicts electropherogram images from various subjects illustrating the variability in size profiles and impact of abstinence time.

[0028] FIG. 17 depicts tissue deconvolution results illustrating that HMW has lower proportion of sperm signal and higher proportion of somatic cell signal in assumed healthy controls.

[0029] FIG.18 depicts tissue deconvolution results for vasectomy subjects in assumed healthy controls.

[0030] FIG.19 depicts unsupervised clustering of reference dataset using our deconvolution signature matrix.

[0031] FIG. 20 depicts values for tissue / cell type specific hypomethylation markers in reference dataset.

[0032] FIG. 21 depicts methylation values for tissue / cell type specific hypomethylation markers in SSD.

[0033] FIG. 22 depicts methylation values for tissue / cell type specific hypomethylation markers in HMW.

[0034] FIG.23 is a table summarizing the reference datasets used for tissue deconvolution (1-4).

[0035] FIG.24 is a table summarizing the signature matrix. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Bodily fluids that contain somatic cells and exDNA include blood, urine, saliva, cerebrospinal fluid (CSF) and semen. While much work has been done to study DNA in blood and urine, there has been less work conducted to analyze DNA in CSF, saliva and semen. While semen samples are typically used to study infertility issues, semen samples may also present an alternative method to analyze prostate-specific disorders, given the role of the prostate and other reproductive organs in the production of seminal fluid, and the resulting somatic cell-derived DNA (e.g., exDNA and / or DNA from healthy or cancerous somatic cells) present in the seminal fluid.

[0037] Previous work has found that significantly more exDNA is present in semen samples obtained from cancer patients as compared with semen samples obtained from healthy patients, however the source of that DNA has not been characterized prior to the disclosures herein. See Ponti et al., Pathol Oncol Res. 2018 Oct;24(4):941-945. Since DNA extracted from seminal plasma contains both high molecular weight DNA and low molecular weight DNA from both sperm cells and somatic cells, there will be background signal resulting from sperm cell DNA when analyzing signal from somatic cell DNA.

[0038] In the same way that white blood cell exDNA impacts the ability to detect tumor exDNA in blood, the background signal from sperm exDNA can impact the ability to detect tumor exDNA (e.g.,Attorney Ref.60974.00008WO01 (FLW-0006-WO) prostate or testicular cancer) detection in semen. Accordingly, in order to properly analyze signal(s) corresponding to somatic cell-derived exDNA in a semen sample, background signal(s) corresponding to exDNA derived from sperm cells in the semen sample must be removed to improve sensitivity and to yield more accurate results.

[0039] Sperm DNA is very tightly packaged into a volume typically 10% or less than that of a somatic cell. This is at least in part due to the fact that histones are largely replaced by smaller, highly basic arginine rich protamines. It is estimated that approximately 85% of sperm DNA is associated with protamines as opposed to histones. See Bench et al. Cytometry. 1996 Apr 1;23(4):263-71. This makes sperm DNA very unique from any other cell type in the human body. Protamine-associated cfDNA derived from sperm cells has a high molecular weight.

[0040] Each protamine P1 molecule binds to 10-11 bp of DNA and protamine P2 binds to a slightly larger segment of DNA of approx. 15 bp. See Balhorn, Genome Biol. 2007; 8(9):227. Protamine molecules bind in the major groove of DNA, neutralizing the phosphodiester backbone of DNA and cause the DNA molecules to coil into toroidal structures, which contain approximately 50,000bp of DNA (Balhorn, Genome Biol. 2007;8(9):227). Upon sperm cell death, this tight packaging, toroidal structure and protein protection likely makes the DNA resistant to nucleases present in the semen, and likely results in protamine-associated sperm cfDNA being significantly larger than that seen with histone-associated cfDNA.

[0041] Additionally, sperm cell structure offers properties that make sperm cells resistant to many of the cell lysis methods that effectively lyse somatic cells. Sperm heads possess a nuclear cap and acrosome which protect the nucleus, and protamines to protect the DNA. Sperm DNA cannot be decondensed in vitro without reducing reagents and sperm nuclei appear to be robust even to sonication (see Perreault et al., J Exp Zool. 1982 Dec 10;224(2):253-7; and Kuretake et al., Biol Reprod. 1996 Oct;55(4):789-95).

[0042] exDNA molecules vary in size ranges depending on their source. As a non-limiting example, exDNA in neutrophil extracellular traps (NETs) may be 1-10kb, but may degrade to mononucleosome size with time. See Pisareva et al., Genome Med 14, 135 (2022). Necrotic cfDNA is generally assumed to be large, typically >10kb. See Jahr et al., Cancer Res.2001 Feb 15;61(4):1659-65; Rostami et al. Cell Rep.2020 Jun 30;31(13):107830.

[0043] cfDNA size ranges from 120bp-220bp, or multiples thereof, with a peak at 167bp; this characteristic peak aligns with the length of DNA wrapped around a single nucleosome, plus a short stretch of approx. 20bp linker DNA bound to a histone (Snyder et al., Cell. 2016 Jan 14;164(1-2):57- 68). Studies have shown that fetal cfDNA is slightly larger, around 143bp (Jiang et al., Trends Genet. 2016;32:360–371) and tumor cfDNA slightly shorter, around 150bp (Lapin et al., J Transl Med. 2018 Nov 6;16(1):300).

[0044] The foregoing discoveries form the foundation upon which the embodiments making up the present application were formed, which are described in greater detail below.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0046] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0047] All references cited throughout the disclosure, including patent applications and publications, are incorporated by reference herein in their entirety.

[0048] Recognizing and taking into account the importance and utility of methods that can provide the analyses described herein, the specification and Appendices A and B describe various embodiments for methods for analyzing somatic cell-derived DNA molecules in a semen sample. I. Definitions

[0049] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0050] In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0051] By “comprising” it is meant that the recited elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the scope of the claim.

[0052] By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0053] By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim.

[0054] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0055] As used herein, the phrases “selected from the group consisting of,” “chosen from,” and the like include mixtures of the specified materials.

[0056] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0057] Except where expressly noted, trademarks are shown in upper case.

[0058] Unless stated otherwise, all percentages, parts, ratios, etc., are by weight.

[0059] When an amount, concentration, or other value or parameter is given as a range, or a list of upper and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper and lower range limits, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range. Further, where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

[0060] Further, unless otherwise explicitly stated to the contrary, when one or multiple ranges or lists of items are provided, this is to be understood as explicitly disclosing any single stated value or item in such range or list, and any combination thereof with any other individual value or item in the same or any other list.

[0061] As used herein, an "embodiment" means that a particular feature, structure or characteristic is included in at least one or more manifestations, examples, or implementations of this invention. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art. Combinations of features of different embodiments are all meant to be within the scope of the invention, without the need for explicitly describing every possible permutation by example. Thus, any of the claimed embodiments can be used in any combination.

[0062] The terms “about” or “comprising essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e.,Attorney Ref.60974.00008WO01 (FLW-0006-WO) the limitations of the measurement system. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition. When the term “about” is used, it is used to mean a certain effect or result can be obtained within a certain tolerance, and the skilled person knows how to obtain the tolerance. When the term "about" is used in describing a value or an endpoint of a range, the disclosure should be understood to include the specific value or end-point referred to. An exemplary variance of the recited value is 10% of the value.

[0063] The terms “exDNA” or “extracellular DNA” refer to DNA that resides outside of cells. ExDNA can originate from either passive or active mechanisms. Passive release mechanisms include cell lysis wherein the cell membrane is disrupted, leading to the release of cellular contents (e.g., DNA) and include programmed cell death (apoptosis), necrosis (programmed necrosis), and some forms of NETosis. Active release is when cells secrete exDNA, for example in extracellular vesicles (EVs), which serve a role in cell-to-cell communications, or in some forms of NETosis. The size of exDNA is dependent on its origin (e.g., exDNA resulting from apoptosis is typically around 167bp and considered low molecular weight, whereas exDNA resulting from necrosis is typically >10,000bp and considered high molecular weight). exDNA also includes DNA that has been actively secreted from cells, as well as circulating cell-free DNA (cfDNA). cfDNA may result from DNA released by cells following cell death (e.g., cell death occurring due to cell lysis, apoptosis, necrosis, etc.). exDNA derived from sperm cells can be nucleosomal or protamine-associated. exDNA derived from somatic cells is nucleosomal, and does not include protamine-associated exDNA.

[0064] The term “NETosis” refers to the release of a type of exDNA that results in the formation of neutrophil extracellular traps (NETs). NETosis is a process where neutrophils release their DNA as exDNA within a network of extracellular fibers. There are two types of NETosis: suicidal NETosis (i.e., where cells die) and vital NETosis (i.e., where cells remain viable). See Vorobjeva et al., Biochemistry Moscow 85, 1178–1190 (2020).

[0065] The term “natural cell lysis” means lysis that has occurred due to a biological process and / or lysis that has occurred as a result of environmental conditions (e.g., temperature, pressure, time, etc.) during storage of a sample for collection, transportation, etc.

[0066] The term “somatic cell derived DNA” refers to DNA originating from a somatic cell, and specifically includes DNA molecules from a lysed somatic cell (e.g., natural cell lysis, lysis resulting exposure to a lysis reagent, etc.), as well as exDNA molecules originating from a somatic cell.

[0067] The term “tissue of origin analysis” refers to a further analysis performed on sequencing analysis results generated for a DNA fraction, in order to determine the proportion(s) of DNA present in the DNA fraction based on tissue cell type.

[0068] Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the presentAttorney Ref.60974.00008WO01 (FLW-0006-WO) specification and viewing the present drawings that various modifications and variations can be made thereto. II. Detailed Description Separation Techniques based on Protamine Packaging of Sperm DNA

[0069] In one aspect, provided herein is a method for enriching a semen sample for a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of nucleosomal exDNA molecules derived from sperm cells.

[0070] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes two centrifugation steps. The first centrifugation step includes centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells, and a first supernatant. In some embodiments, the first centrifugation step may include centrifuging the semen sample at about 300- 500g for about 10-20 minutes. As a non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample for 15 minutes. As a further non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample at 400g. The second centrifugation step includes centrifuging the first supernatant to generate a second pellet comprising a plurality of cellular debris and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and a second supernatant comprising the plurality of nucleosomal exDNA molecules derived from sperm cells. In some embodiments, the second centrifugation step may include centrifuging the first supernatant at about 10,000-18,000g for about 7- 15 minutes. As a non-limiting example, in some embodiments the second centrifugation step may include centrifuging the first supernatant for 10 minutes. As a further non-limiting example, in some embodiments the second centrifugation step may include centrifuging the semen sample at 16,000g. In some embodiments, the second supernatant can be isolated from the second pellet after the second centrifugation step.

[0071] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes one centrifugation step. In some embodiments, the centrifugation step includes centrifuging the semen sample to generate a pellet comprising a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and a supernatant comprising the plurality of nucleosomal exDNA molecules derived from sperm cells. In some embodiments, the centrifugation step may include centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments the centrifugation step may include centrifuging the semen sample for 10 minutes. As a further non-limiting example, in some embodiments the centrifugation step mayAttorney Ref.60974.00008WO01 (FLW-0006-WO) include centrifuging the semen sample at 16,000g. In some embodiments, the supernatant can be isolated from the pellet after the centrifugation step.

[0072] In some embodiments, a size-based separation procedure may be performed on the DNA fraction. Size-based separation procedures are discussed in further detail below.

[0073] In another aspect, provided herein is a method for enriching a semen sample for a plurality of exDNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of exDNA molecules derived from somatic cells and a plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of exDNA molecules derived from somatic cells.

[0074] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes two centrifugation steps. The first centrifugation step includes centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant. In some embodiments, the first centrifugation step may include centrifuging the semen sample at about 300-500g for about 10-20 minutes. As a non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample for 15 minutes. As a further non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample at 400g. The second centrifugation step includes centrifuging the first supernatant to generate a second pellet comprising a plurality of cellular debris and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and a second supernatant comprising the plurality of exDNA molecules derived from somatic cells. In some embodiments, the second centrifugation step may include centrifuging the first supernatant at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments the second centrifugation step may include centrifuging the first supernatant for 10 minutes. As a further non- limiting example, in some embodiments the second centrifugation step may include centrifuging the first supernatant at 16,000g. In some embodiments, the second supernatant can be isolated from the second pellet after the second centrifugation step.

[0075] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes one centrifugation step. In some embodiments, the centrifugation step includes centrifuging the semen sample to generate a pellet comprising a plurality of intact sperm cells and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and a supernatant comprising the plurality of exDNA molecules derived from somatic cells. In some embodiments, the centrifugation step may include centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments the centrifugation step may include centrifuging the semen sample for 10 minutes. As a further non-limiting example, in some embodiments the centrifugation step may include centrifuging the semen sample at 16,000g. In some embodiments, the supernatant can be isolated from the pellet after the centrifugation step.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0076] In some embodiments, a size-based separation procedure may be performed on the DNA fraction. Size-based separation procedures are discussed in further detail below.

[0077] In some embodiments, the plurality of exDNA molecules derived from sperm cells in the semen sample may be analyzed.

[0078] In some embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules secreted by somatic cells in the semen sample. In some embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample. As a non-limiting example, somatic cells in the semen sample that have undergone natural cell lysis can include somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0079] In one or more embodiments, somatic cells in the semen sample include prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof. Differential Lysis of Sperm Cells and Somatic Cells

[0080] In one aspect, provided herein is a method for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules from the somatic cells; centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

[0081] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes two centrifugation steps. The first centrifugation step includes centrifuging the semen sample to generate a first pellet and a first supernatant. In some embodiments, the first pellet includes a plurality of intact sperm cells, a plurality of intact somatic cells, or any combination thereof. In some embodiments, the first centrifugation step may include centrifuging the semen sample at about 300- 500g for about 10-20 minutes. As a non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample for 15 minutes. As a further non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample at 400g.

[0082] The second centrifugation step includes centrifuging the first supernatant to generate a second pellet and a second supernatant. In some embodiments, the second pellet includes a plurality of intact sperm cells, a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine- associated exDNA molecules derived from sperm cells, or any combination thereof. In some embodiments, the second centrifugation step may include centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments the secondAttorney Ref.60974.00008WO01 (FLW-0006-WO) centrifugation step may include centrifuging the semen sample for 10 minutes. As a further non-limiting example, in some embodiments the second centrifugation step may include centrifuging the semen sample at 16,000g. In some embodiments, the second supernatant can be isolated from the second pellet after the second centrifugation step.

[0083] In some embodiments, centrifuging the semen sample to generate the DNA fraction includes one centrifugation step. In some embodiments, the centrifugation step includes centrifuging the semen sample to generate a pellet and a supernatant. In some embodiments, the pellet includes a plurality of intact sperm cells, a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, or any combination thereof. In some embodiments, the centrifugation step may include centrifuging the semen sample at about 10,000- 18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments the centrifugation step may include centrifuging the semen sample for 10 minutes. As a further non-limiting example, in some embodiments the centrifugation step may include centrifuging the semen sample at 16,000g. In some embodiments, the supernatant can be isolated from the pellet after the centrifugation step.

[0084] In one or more embodiments, cellular debris from sperm cells that have undergone natural cell lysis includes cellular debris from sperm cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample. In some embodiments, the plurality of somatic cells in the semen sample that have undergone natural cell lysis include somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0085] In one or more embodiments, cellular debris from lysed somatic cells includes a plurality of somatic cells in the semen sample that have undergone natural cell lysis, a plurality of somatic cells in the semen sample selectively lysed by the lysis reagent, or any combination thereof.

[0086] In one or more embodiments, somatic cells in the semen sample include prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof.

[0087] In one or more embodiments, the DNA fraction further includes a plurality of exDNA molecules derived from sperm cells in the semen sample. In one or more embodiments, the DNA fraction further includes a plurality of exDNA molecules derived from somatic cells in the semen sample. As a non-limiting example, the plurality of exDNA molecules derived from somatic cells in the semen sample includes a plurality of exDNA molecules secreted by somatic cells in the semen sample. As a further non-limiting example, the plurality of exDNA molecules derived from somatic cells in the semen sample comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample. In some embodiments, somatic cells in the semen sample that have undergone natural cell lysis include somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0088] In another aspect, provided herein is a method for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules from the somatic cells and the somatic cell lysis reagent; centrifuging the mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

[0089] In some embodiments, the method further comprises isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; and isolating the third supernatant from the third pellet, thereby enriching the sample for the plurality of exDNA molecules derived from somatic cells in the semen sample. In some embodiments, the plurality of exDNA molecules derived from somatic cells in the semen includes a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample. In some embodiments, somatic cells in the semen sample that have undergone natural cell lysis includes somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0090] In some embodiments, the first pellet and the first supernatant are generated by centrifuging the semen sample at about 300-500g for about 10-20 minutes. As a non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample for 15 minutes. As a further non-limiting example, in some embodiments the first centrifugation step may include centrifuging the semen sample at 400g. In some embodiments, the first pellet can include a plurality of intact sperm cells, a plurality of intact somatic cells, or any combination thereof.

[0091] In some embodiments, the second pellet and the second supernatant are generated by centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments, generation of the second pellet and the second supernatant can include centrifuging the first supernatant for 10 minutes. As a further non-limiting example, in some embodiments, generation of the second pellet and the second supernatant can include centrifuging the first supernatant at 16,000g.

[0092] In some embodiments, the second pellet includes a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, or any combination thereof. In some embodiments, cellular debris from sperm cells that have undergone natural cell lysis includes cellular debris from sperm cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semenAttorney Ref.60974.00008WO01 (FLW-0006-WO) sample. In some embodiments, cellular debris from lysed somatic cells includes a plurality of somatic cells in the semen sample that have undergone natural cell lysis, a plurality of somatic cells in the semen sample selectively lysed by the lysis reagent, or any combination thereof. In some embodiments, the plurality of somatic cells in the semen sample that have undergone natural cell lysis includes somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0093] In some embodiments, the third pellet and the third supernatant are generated by centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes. As a non-limiting example, in some embodiments, generation of the third pellet and the third supernatant can include centrifuging the first supernatant for 10 minutes. As a further non-limiting example, in some embodiments, generation of the third pellet and the third supernatant can include centrifuging the first supernatant at 16,000g.

[0094] In one or more embodiments, somatic cells in the semen sample include prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof.

[0095] In one or more embodiments, a method as described herein further includes analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

[0096] In some embodiments, methods as described herein include intentional lysing of somatic cells using a lysis reagent that is designed to take advantage of differential lysis characteristics of sperm cells as compared to somatic cells (e.g., using a lysis reagent capable of lysing only somatic cells, while not affecting sperm cells). Quantitative Analysis of Somatic Cell-Derived DNA in a Semen Sample

[0097] In one aspect, provided herein is a method for analyzing a quantity of somatic cell derived DNA molecules from a specific somatic cell type in a semen sample, the method comprising: isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the DNA fraction to determine an abundance of somatic cell derived DNA molecules from the specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a sufficient quantity of the somatic cell derived DNA molecules from the specific somatic cell type for analysis of a prostate-specific disorder. In some embodiments, the sequencing analysis may be performed using any known methods, such as, for example without limitation, a methylation analysis.

[0098] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the secondAttorney Ref.60974.00008WO01 (FLW-0006-WO) supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0099] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a pellet and a supernatant; and isolating the supernatant from the pellet, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, wherein the supernatant comprises the DNA fraction, and wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0100] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the plurality of somatic cells; centrifuging the semen sample to generate a first pellet and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from the plurality of somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0101] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the somatic cells; centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells; and isolating the supernatant from the pellet, wherein the supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules from the somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0102] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules derived from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules derived from the somatic cells and the somatic cell lysis reagent; centrifuging theAttorney Ref.60974.00008WO01 (FLW-0006-WO) mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells. In some embodiments, the method described herein further comprises: isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; and isolating the third supernatant from the third pellet, wherein the third supernatant comprises a second DNA fraction that comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells.

[0103] In one or more embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample. In one or more embodiments, the somatic cells in the semen sample that have undergone natural cell lysis include somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0104] In one or more embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules secreted by somatic cells in the semen sample.

[0105] In some embodiments, the methods as described herein further include analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

[0106] In some embodiments, the specific somatic cell type and / or the plurality of somatic cells include prostate cells, prostate cancer cells, white blood cells, testicular cells, kidney cells, bladder cells, or any combination thereof.

[0107] In some embodiments, the prostate-specific disorder is prostate cancer or testicular cancer. In some embodiments, the prostate-specific disorder is benign prostatic hyperplasia (BPH). In some embodiments, the prostate-specific disorder is prostatitis.

[0108] In some embodiments, a size-based separation procedure may be performed on the DNA fraction. Size-based separation procedures are discussed in further detail below.

[0109] In some embodiments, comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a sufficient quantity of the somatic cell derived DNA molecules from the specific somatic cell type for analysis of a prostate-specific disorder includes performing a tissue of origin analysis. In some embodiments, a tissue of origin analysis may be, as a non-limiting example, a tissue deconvolution analysis.Attorney Ref.60974.00008WO01 (FLW-0006-WO) Detection of a Prostate-Specific Disorder Through Analysis of Somatic Cell Derived DNA in a Semen Sample

[0110] In one aspect, provided herein is a diagnostic method for detecting a prostate-specific disorder from a semen sample, the method comprising: isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the plurality of somatic cell derived DNA molecules to determine an abundance of somatic cell derived DNA molecules from a specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules derived from cells of the specific somatic cell type to a threshold value to determine whether the semen sample contains a clinically relevant prostate-specific disorder signal. In some embodiments, the sequencing analysis may be performed using any known methods, such as, for example without limitation, a methylation analysis.

[0111] In some embodiments, the diagnostic method as described herein may be conducted following identification of a predetermined quantity of somatic cell derived DNA molecules from a specific somatic cell type in a semen sample, as described herein.

[0112] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0113] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a pellet and a supernatant; and isolating the supernatant from the pellet, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, wherein the supernatant comprises the DNA fraction, and wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0114] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the plurality of somatic cells; centrifuging the semen sample to generate a first pellet and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet and a second supernatant; and isolating the secondAttorney Ref.60974.00008WO01 (FLW-0006-WO) supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from the plurality of somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

[0115] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the somatic cells; centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells; and isolating the supernatant from the pellet, wherein the supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules from the somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample. In some embodiments, the method mentioned herein further comprises:

[0116] In some embodiments, isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules derived from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules derived from the somatic cells and the somatic cell lysis reagent; centrifuging the mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells. In some embodiments, the method described herein further comprises: isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; and isolating the third supernatant from the third pellet, wherein the third supernatant comprises a second DNA fraction that comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells.

[0117] In one or more embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample. In one or more embodiments, the somatic cells in the semen sample that have undergone natural cell lysis include somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

[0118] In one or more embodiments, the plurality of exDNA molecules derived from somatic cells includes a plurality of exDNA molecules secreted by somatic cells in the semen sample.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0119] In some embodiments, the diagnostic methods as described herein further include analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

[0120] In some embodiments, the specific somatic cell type and / or the plurality of somatic cells include prostate cells, prostate cancer cells, white blood cells, testicular cells, kidney cells, bladder cells, or any combination thereof.

[0121] In some embodiments, the prostate-specific disorder is prostate cancer or testicular cancer. In some embodiments, the prostate-specific disorder is benign prostatic hyperplasia (BPH). In some embodiments, the prostate-specific disorder is prostatitis.

[0122] In some embodiments, a size-based separation procedure may be performed on the DNA fraction. Size-based separation procedures are discussed in further detail below.

[0123] In some embodiments, comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a clinically relevant prostate-specific disorder signal comprises performing a tissue of origin analysis. Size-Based Separation Procedure

[0124] In one or more embodiments, methods as described herein further comprise performing a size- based separation procedure on the DNA fraction. In some embodiments, performing the size-based separation procedure comprises separating the DNA fraction into a low molecular weight fraction, a high molecular weight fraction, or a combination thereof. In some embodiments, the low molecular weight fraction comprises molecules (e.g., exDNA, somatic cell derived DNA molecules, or a combination thereof having a size smaller than 500bp. In some embodiments, the high molecular weight fraction comprises molecules (e.g., exDNA, somatic cell derived DNA molecules, or a combination thereof) having a size greater than or equal to 500bp.

[0125] In some embodiments, a size-based separation procedure is performed on the high molecular weight fraction as described herein, to generate a second low molecular weight fraction comprising molecules (e.g., exDNA, somatic cell derived DNA molecules, or a combination thereof) having a size smaller than 800bp, and a second high molecular weight fraction comprising molecules (e.g., exDNA, somatic cell derived DNA molecules, or a combination thereof) having a size greater than or equal to 800bp.

[0126] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. Semen Sample

[0127] In some embodiments, a semen sample is processed from a patient to generate the seminal plasma sample. In some embodiments, the patient from which the semen sample is obtained has abstained from ejaculation for a time period ranging from 2 to 5 days (e.g., for at least 2 days, for atAttorney Ref.60974.00008WO01 (FLW-0006-WO) least 2.5 days, for at least 3 days, for at least 3.5 days, for at least 4 days, for at least 4. 5 days, for 2 days, for 3 days, for 4 days, for 5 days, for 2.5 to 4.5 days, or for 3 to 4 days,).

[0128] In some embodiments, the semen sample is processed within 12 hours, within 9 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1.5 hours, within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes, or immediately after / of donation. Between donation and processing, the semen sample is stored at room temperature (i.e., 20-22°C) or lower, for example at 4°C to 15°C, at 4°C to 12°C, at 4°C to 8°C.

[0129] In some embodiments, the semen sample may be processed after extended storage at 0°C or lower, -10°C or lower, -20°C or lower, -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, -70°C or lower, -80°C or lower. As a lower limit, -90°C is mentioned. It is further envisioned that the storage temperature may be within a range embraced by any of the foregoing recited extended storage temperatures as the upper limit and -90°C as the lower limit. Other extended storage temperature ranges are also envisioned including 0°C to -80°C, 0°C to -70°C, 0°C to -60°C, 0°C to -50°C, 0°C to -40°C, 0°C to -30°C, 0°C to -20°C, 0°C to -10°C, -10°C to -80°C, -10°C to -70°C, -10°C to -60°C, -10°C to - 50°C, -10°C to -40°C, -10°C to -30°C, -10°C to -20°C, -20°C to -80°C, -20°C to -70°C, -20°C to -60°C, -20°C to -50°C, -20°C to -40°C, and -20°C to -30°C. Wherein when the semen sample is stored at 0°C or lower, the sample is stored in a semen sample preservative solution (infra) such that the sperm cells do not lyse and / or the proteins do not denature. In some embodiments, the semen sample is subjected to a fixation process prior to freezing. In some embodiments, the semen sample may be cryopreserved.

[0130] Where the semen sample is stored for an extended period (e.g., a time greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours to less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days) the semen sample may be admixed with a semen sample preservative. Exemplary semen sample preservatives include cryoprotectants (e.g., dimethyl sulfoxide (DMSO), dimethylformamide (DMF), glycerol, and propylene glycol), formaldehyde, a formaldehyde-derivative, or semen extenders. Exemplary formaldehyde- derivatives include Quaternium-15, DMDM hydantoin, methenamine, methanediol, benzylhemiformal, urea-formaldehyde, urea, paraformaldehyde, pentaerythritol, imidazolidinyl urea, diazolidinyl urea, polyoxymethylene urea, sodium hydroxymethylglycinate, 2-bromo-2-nitropropane-1,3-diol (bronopol), bronidox, and glyoxal.

[0131] Semen extenders include those developed to protect sperm, conserve motility, and fertility over time by stabilizing the plasmalemma, provide energy substrates, and prevent harmful effects of pH and osmolarity changes. The use of semen extenders has the advantage of preserving the sperm in a living state for an extended period, which avoids DNA degradation caused by nucleases present in the semen. Examples of components of semen extenders include low-density lipoprotein and lecithin. In some embodiments, the preservation fluid or sperm preservation fluid may comprise pH stabilizing agents and ions and nutrients to promote sperm health. In some embodiments, the preservation fluid may comprise salts, sugar, and / or an antibiotic agent.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0132] In some embodiments, the semen sample as used in the methods described herein can be contacted with a preservative. In some embodiments, the semen to preservative ratio can range from about 0.1 to about 16 prior to centrifugation. In some embodiments, the methods described herein can be conducted without freezing the sperm sample.

[0133] In some embodiments, methods described herein can be conducted without freezing the semen sample.

[0134] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

[0135] Although this invention has been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.

[0136] Various modifications to the embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others or ordinary skill in the art to understand the embodiments disclosed herein. Thus, this invention is not intended to be limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and features disclosed herein. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

[0137] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Materials and Methods

[0138] An IRB approved study, “Fellow Reproductive Improvement Study for Knowledge and Excellence (FRISKE)”, enrolled healthy participants to provide fresh semen samples. Preservative was either added immediately or within 3 hours of collection.

[0139] An IRB approved study, “Prostate Cancer Biomarker Detection in Seminal Plasma (PRISM)” enrolled men scheduled to undergo a prostate biopsy. A collection kit (with preservative) was mailed to their home and prior to undergoing biopsy, a semen sample was collected and shipped back to Applicant for use as a clinical cohort. Upon receiving biopsy results, clinical data was entered into the electronic data capture (EDC). Twenty samples were processed as part of a “run-in” and data included here (note: biopsy data was not received from one patient, so only 19 patients are represented).Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0140] 500ul of preservative was added to 500ul-6ml semen, and stored for 1-7 days (most experiments used samples stored for 2 days). Seminal plasma was prepared by centrifuging at 400g for 15 mins, transferring supernatant to a new tube and performing a second spin at 16,000g for 10 mins. Alternatively, seminal plasma was prepared by performing a single 16,000g spin for 10 mins. exDNA was extracted from seminal plasma following the Qiagen™ Circulating Nucleic Acids Extraction kit instructions using 1-3ml seminal plasma. High molecular weight (>800bp) and small molecular weight DNA (<500bp) were separated as follows: a double size selection was performed using a 0.6x / 1.8x ratio of SPRI reagent to sample volume. The high molecular weight DNA was eluted from the beads and underwent a further size selection using 0.5x ratio of SPRI reagent to sample volume. The high molecular weight DNA was subject to sonication using a Covaris™ ultrasonicator, followed by a SPRI clean up using 1.5x ratio of SPRI reagent to sample volume.

[0141] Methylation libraries were then prepared for both the small size selected DNA and the high molecular weight DNA using New England Biolab™ NEBNext® Enyzmatic Methyl-Seq kit. Target enrichment was then performed using the Twist™ Human Methylome Panel. Next Generation Sequencing was performed on a NovaSeqX (either on a 10B or 25B flowcell) followed by further analyses (e.g., tissue of origin analysis, such as tissue deconvolution). III. Examples Example 1: Tissue of Origin Analysis with Low Molecular Weight DNA from Seminal Fluid from Healthy Controls

[0142] FIG. 1 shows the results of a tissue deconvolution analysis of semen samples from assumed healthy individuals, revealing the proportion of small molecular weight DNA signal 2 days after collection, as a function of the tissue from which the cell signal was received. The largest signal is from sperm, with the exception of a subject who had undergone a vasectomy. The largest somatic cell signal is from blood (i.e., a white blood cell signal) or prostate. For example, one non-vasectomy subject had 20% prostate signal, which is higher than any white blood cell (WBC) signal in a non-vasectomy subject. Example 2: Tissue of Origin Analysis with High Molecular Weight DNA from Seminal Fluid from Healthy Controls

[0143] FIG. 2 shows the results of a tissue deconvolution analysis of semen samples from healthy individuals, revealing the proportion of high molecular weight DNA signal 2 days after collection, as a function of the tissue from which the cell signal was received. The largest signal is from sperm, though it is vastly reduced as compared to the low molecular weight DNA sperm signal seen in FIG. 1. The largest somatic cell signal is from blood (i.e., a white blood cell signal) or prostate (for example, in some subjects, prostate signal is higher than WBC); however when compared to the somatic cell signals in FIG.1, all somatic cell signals in FIG.2 are increased.Attorney Ref.60974.00008WO01 (FLW-0006-WO) Example 3: Tissue of Origin Analysis with High Molecular Weight DNA from Seminal Fluid from “run-in” PRISM samples

[0144] FIG.3 shows the results of a tissue deconvolution analysis of “run-in” PRISM semen samples, revealing the proportion of high molecular weight DNA signal as a function of the tissue from which the cell signal was received by vasectomy status. In these samples from patients with varying prostate conditions (cancer, BPH, prostatitis), a much larger signal from white blood cells is seen, relative to the signal seen from samples from healthy individuals in FIG. 2 (discussed in Example 2). This suggests that irrespective of the underlying condition, there is a strong immune component. Example 4: Difference in signal between High Molecular Weight (HMW) DNA and Small Size Selected (SSD) DNA in the clinical cohort and healthy controls

[0145] FIG. 4 shows the results of a methylated tissue deconvolution analysis, and the difference between signals received from high molecular weight (HMW) DNA and small size selected (SSD) DNA in the run in for the PRISM clinical cohort and healthy controls. The results in FIG.4 reveal enrichment of somatic cell signal (e.g., from prostate cells, testicular cells, white blood cells, etc.) and a decrease in sperm cell signal, when analysis is performed on the HMW fraction versus the SSD fraction. Example 5: Removal of background noise caused by sperm DNA by introducing a high-speed second spin

[0146] FIGS.5A-6 show the results of electrophoresis analyses (Tapestation high sensitivity dsDNA) and tissue deconvolution analysis, respectively, performed on samples from vasectomy patients (FIG. 5A) and non-vasectomy patients (FIG. 5B). The electrophoresis analyses show sample intensity with normalized fluorescent units (FU) with respect to size of the DNA (bp) of the following conditions in a semen sample: with a 16,000g second spin, with a 10,000g second spin, or with no second spin. Generally, there appeared to be no significant difference between a second spin at 10,000g or 16,000g. However, eliminating the second spin resulted in a huge amount of additional DNA in the samples from non-vasectomy subjects, while having no impact in the samples from vasectomy subjects. These results suggest that the second spin can remove sperm DNA, and thus, background noise caused by sperm DNA could potentially be removed by performing a high-speed spin (approximately 10,000-16,000g, and where at least 10,000g could be sufficient to remove sperm DNA).

[0147] FIG.6 shows the results from tissue deconvolution analysis of the samples tested in FIGS.5A- 5B, confirming that sperm is the source of the additional DNA present when a second spin is not performed. Example 6: Single spin of semen samples as compared to two spins

[0148] FIG.7 shows the results of tissue deconvolution analyses comparing the results of performing only a single spin, and performing two spins with the speed of the second spin being at various speeds.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0149] These results suggest that eliminating the first, lower speed (approximately 400g) spin and only performing a single higher speed spin can be done without impacting tissue deconvolution results. These results also show that, if two spins are performed, the second spin could be done at a speed of approximately 3,000g to 16,000g. Generally, the speed of the second, “high speed” spin could be as low as 3,000g and still provide the accuracy provided by the higher speeds of 10,000g or 16,000g. Example 7: Tissue deconvolution data of cell signals from non-vasectomy patients in HMW and SSD

[0150] FIG. 8 shows the results of cell signals from the full PRISM clinical study cohort, from non- vasectomy patients. These results show additional tissue deconvolution data supporting the claim that somatic cell signal in non-vasectomy subjects can be enriched by selecting for HMW DNA. These plots show lower sperm signal and higher somatic cell signal in HMW than SSD in PRISM clinical studies, wherein n = 150. Participants in PRISM represent the patient population for the non-limiting intended use of the methods disclosed herein, which include patients scheduled to undergo a prostate biopsy. In these results, the HMW is much more enriched for granulocytes, rather than prostate, but the results for the prostate still show higher in HMW than in SSD.

[0151] FIG. 9 is a table showing the values of actual percentage differences between the HMW and SSD for each tissue type within the PRISM study samples shown in FIG. 8. As noted in the table, the HMW DNA has less sperm signal (over 17% less) and more somatic cell signal than SSD. Example 8: Exemplary methods for analyzing somatic cell-derived DNA molecules in a semen sample

[0152] Provided herein are non-limiting examples for carrying out any of the methods disclosed herein.

[0153] FIGS. 10A-10B summarize an exemplary method for sperm sample analysis. FIG. 10A is a diagrammatic flow chart showing isolation of DNA from a semen sample. In some embodiments, a semen sample can be stored in a receptable, transferred to a tube, and centrifuged according to the flow chart. FIG. 10B shows exemplary data obtained from a DNA isolated from a semen sample according to the methods disclosed herein. cfDNA can be extracted from a sample to show low MW DNA, high MW DNA, and size selection can also be performed for more refined results. Example 9: Comparison of men sent for prostate biopsy compared to healthy controls in HMW

[0154] FIG. 11 summarizes the results of analyses performed using samples from a PRISM study as compared to a FRISKE study. These results from high molecular weight DNA show that there is a much higher immune component in PRISM clinical study samples compared to FRISKE study samples, which served as healthy controls.Attorney Ref.60974.00008WO01 (FLW-0006-WO) Example 10: Tissue deconvolution analyses of size selected small cfDNA and high molecular weight cfDNA

[0155] FIG. 12 summarizes the results of tissue deconvolution analysis of size selected small cfDNA (SSD). These results show a strong immune component in both cancer and non-cancer PRISM samples.

[0156] FIG. 13 summarizes the results of tissue deconvolution analysis of high molecular weight cfDNA (HMW). These results show a strong immune component in both cancer and non-cancer PRISM samples. Example 11: Tissue of origin characterization of cell free DNA in seminal plasma: implications for new liquid biopsies

[0157] Provided herein are disclosures relating to characterization of cfDNA in seminal fluid through tissue of origin studies, and study of seminal fluid as a potential tool for liquid biopsy. Abstract and Significance Summary

[0158] Liquid biopsies are becoming increasingly used for the detection and monitoring of disease states (1–3). While cell free DNA (cfDNA) in blood and urine have been well studied, much less is known about the composition of cfDNA in seminal fluid. We sought to characterize cfDNA in seminal fluid through tissue of origin studies using methylation analysis in men aged 21-60 yrs. We confirmed that seminal fluid contains an abundance of cfDNA that is both nucleosomal and >1 kb (4). However, here we demonstrate for the first time that the high molecular weight (HMW) DNA harbors a lower sperm signal and higher somatic cell signal compared to the nucleosomal fraction. Prostate, granulocytes and kidney showed a mean predicted increased contribution of 6.2%, 4.9% and 2.9%, respectively in the HMW fraction. While sperm was the predominant signal in most men without vasectomies, the proportion of predicted prostate contribution reached as high as 26.5% in the HMW fraction. In other subjects without vasectomies, granulocyte cfDNA made up most of the signal. We also observed subject-specific cfDNA size distribution patterns that were reproducible over time, irrespective of abstinence times. These results suggest that seminal fluid is a rich source of cfDNA from various somatic cell types, and enriching for the HMW fraction would yield even higher sensitivity for somatic cfDNA detection. Considering these novel findings, it appears that seminal fluid could serve as liquid biopsy for the detection and monitoring of prostate cancer, benign prostate hyperplasia, prostatitis and infertility.

[0159] While cell free DNA (cfDNA) has been found to be abundant in seminal fluid, its tissue of origin has not been well studied. We developed a tissue deconvolution algorithm to estimate the proportions of different cell types present. We discovered that seminal fluid contains cfDNA from a range of cell types, not just sperm. Moreover, we discovered that the high molecular weight fraction, usually thought of as noise in blood based liquid biopsies, has higher levels of somatic cell signal compared to the nucleosomal fraction. Seminal fluid therefore has the potential to be used as a liquidAttorney Ref.60974.00008WO01 (FLW-0006-WO) biopsy for the detection and monitoring of prostate cancer, benign prostate hyperplasia, prostatitis and infertility. Introduction

[0160] Seminal fluid is a complex mixture of secretions from the seminal gland, prostate, testes, epididymis, and bulbourethral glands. It not only serves as a medium for sperm transport but also provides nutrients and a protective environment for spermatozoa during their journey through the female reproductive tract. Seminal fluid contains a complex range of organic and inorganic constituents, including proteins, enzymes, lipids, carbohydrates, and various ions, which play crucial roles in sperm motility and viability. Despite the emerging interest in liquid biopsies in cancer diagnostics and beyond, seminal fluid has not been extensively studied as a potential specimen type. Here we characterize the composition of cell-free DNA (cfDNA) in seminal fluid, highlighting its potential use in the liquid biopsy space.

[0161] Extracellular DNA (exDNA) or cfDNA is thought to be released into the extracellular environment through apoptosis, necrosis, NETosis, or active secretion by cells (5,6). Given the abundance of sperm in semen, one could expect the large majority of cfDNA in seminal fluid to arise from apoptosis of sperm cells either undergoing spermatogenesis in close proximity to the seminiferous tubule lumen, or during storage of mature sperm in the epididymis. Such cfDNA could serve as biomarkers for fertility as reported by Chou et al. (7), whereby they demonstrated correlations between cfDNA size and important sperm parameters, and Di Pizio et al. (8) who found significantly higher cfDNA levels in patients with sperm abnormalities compared to controls. Other sources of cfDNA in semen could arise from the prostate, bladder, kidney, reproductive tract cells, or resident immune cells. One group proposed seminal plasma cfDNA as a potential biomarker for prostate cancer given their observation of significantly higher concentrations of cfDNA in prostate cancer compared to controls, and larger cfDNA fragment sizes in prostate cancer patients compared to those with benign prostate hyperplasia (BPH) and healthy controls (4,9,10). In their study on cfDNA levels and its association with sperm abnormalities, Di Pizio et al. (8) concluded that it may be of interest to study the cfDNA’s origin and clearance and its methylation profile. One group showed LGALS3 cfDNA methylation status in seminal fluid to be able to discriminate between prostate cancer and benign prostate hyperplasia (11), but to date, no-one to our knowledge has fully characterized the cfDNA methylation signatures present in seminal fluid to understand the relative abundance of cfDNA from the various cell / tissue types that make up the male genitourinary system. Moreover, to our knowledge, no-one has characterized the cell / tissue of origin of the various cfDNA fragment lengths that have previously been correlated with prostate cancer (4). We sought to further investigate these findings by characterizing cfDNA yield, fragment size, and cell / tissue of origin from healthy volunteers. Once the composition of cfDNA in seminal fluid is better understood, we can begin to think about its potential use as a liquid biopsy for fertility, prostate cancer, testicular cancer, benign prostate hyperplasia and prostatitis.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0162] Finally, liquid biopsy test manufacturers working with blood and urine have adopted the use of various preservatives to stabilize cells and nucleic acids so samples can be shipped without fear of losing sensitivity (12,13). If blood is collected without the use of a suitable preservative, cell free nucleic acids will degrade, and white blood cells will die and lyse releasing high molecular weight DNA. This will result in a massive amount of background signal that would likely mask the signal of the already low analyte of interest. To date however, there are no described preservatives that have been shown to stabilize sperm and nucleic acids in seminal fluid. In this study we also sought to identify such a preservative. Example 11 Results Stabilization of Sperm / cfDNA

[0163] FIG.14 depicts electropherogram images illustrating the stabilization of sperm and cfDNA with preservation solution. x-axis is size (bp) and y-axis is signal intensity. A: Comparison of the same sample with and without preservative on Day 0 (seminal fluid processed within 2 hrs of collection). B: Sample with preservative on Day 0 and Day 3 in the oven. C: Sample without preservative on Day 0 and Day 3 in the oven.

[0164] To enable a semen sample to be produced at home as a liquid biopsy, it would be necessary to use a preservative that stabilizes sperm in addition to maintaining the integrity of the cfDNA during shipment to the laboratory. In this study we identified a preservative that could successfully achieve this. FIG.14 illustrates a comparison of the same sample with or without preservative. FIG.14A shows that on Day 0 the profiles look largely the same (both were processed within 2 hours of collection). At Day 3 in the oven (with oscillating temperatures from 10°C- 35°C), the sample with preservative maintains a very similar profile (FIG.14B), while the one without preservative shows evidence of cell lysis and a massive increase in the amount of DNA recovered (FIG.14C). Note that when cfDNA was extracted from seminal fluid without preservative but within 2 hours of collection, we usually observed nucleosomal peaks (~150-200 bp) in addition to a peak >1 kb (FIG.14A). However, in some individuals, only the peak >1 kb was observed (see cfDNA size distribution profile section below). cfDNA Yield

[0165] FIG. 15 depicts cfDNA yields correlated weakly with volume. A: Total yield (ng) by volume of seminal plasma (SP) into extraction. B: Normalized yield (ng per ml SP) by volume of seminal fluid (SF).

[0166] Similar to other studies, we found the cfDNA yield in seminal plasma to be very high. The average total yield was 1.2 ug and there was a weak correlation between total yield and the volume of seminal plasma used in DNA extraction (FIG.15A). When normalized to the volume of seminal plasma, the average was 1003 ng per ml seminal plasma, which is at least an order of magnitude higher thanAttorney Ref.60974.00008WO01 (FLW-0006-WO) that seen in blood plasma or urine. Values ranged from 260 ng - 3229 ng per ml seminal plasma (FIG. 15B). cfDNA Size Distribution Profile by Electrophoresis

[0167] FIG. 16 depicts electropherogram images from various subjects illustrating the variability in size profiles and impact of abstinence time. A: Example of a participant with two dominant cfDNA peaks, one nucleosomal cfDNA peak and one >1 kb. B: Example of a participant with what appears to be multi-nucleosomal cfDNA peaks and one peak >1 kb. C: Example of a vasectomy participant just one peak >1 kb. While these profiles were typically seen in subjects having undergone vasectomy, they were also seen occasionally in subjects not having undergone vasectomy. D: Three different seminal fluid cfDNA samples from the same participant after either 1, 3 or 6 days of abstinence. E: Two different seminal fluid cfDNA samples from the same patient after either 1 or 2 days of abstinence.

[0168] In this study we observed several different characteristic cfDNA size-distribution profiles that appear to be subject specific and may be associated with interesting underlying biology. In most cases the predominant nucleosomal peak appeared to be closer to 200 bp by Tapestation analysis (see FIG. 16A) rather than the typical 166 bp characteristic of nucleosomal cfDNA, but upon sequencing, the mean peak insert size was 146 bp. In some cases, there was a single clean nucleosomal peak (FIG.16A), while others had clear multi-nucleosomal peaks (FIG. 16B) along with the >1 kb peak. In the case of vasectomy participants, we observed no prominent distinct nucleosomal cfDNA peak, but only the peak >1 kb (FIG. 16C). Given the absence of an abundant distinct peak of small (nucleosomal) cfDNA in men having undergone vasectomy, this suggests that most of the small cfDNA is coming from sperm. It is worth noting that in every participant, including those with vasectomies, there was a peak >1 kb. This was of great interest as it indicated that the cfDNA >1 kb was derived, at least in part, from somatic cells. To investigate whether the variability between subjects could also be associated with abstinence times, two subjects provided samples after various periods of abstinence. As can be seen in Figures 3D, abstinence time did not appear to be a factor in the overall profile, but did seem to impact the total amount of cfDNA. After 6 days of abstinence the yield was 2297 ng / ml plasma as compared to only 989 ng / ml plasma after 1 day of abstinence. However, the percent nucleosomal DNA, (i.e. between 50 and 700 bp as measured by Tapestation), remained at approximately 50%. FIG. 16E represents the cfDNA profile from a different subject abstaining for either 1 day or 2 days. In this case, there was no difference in either the cfDNA profile or the yield. The images in Figures 3D and 3E also illustrate the often unique, but reproducible, cfDNA size-distribution profiles from the same subject. Methylation Profile

[0169] FIG. 17 depicts tissue deconvolution results illustrating that HMW has lower proportion of sperm signal and higher proportion of somatic cell signal. A: Tissue deconvolution results for SSDAttorney Ref.60974.00008WO01 (FLW-0006-WO) (n=15). B: Tissue deconvolution results for HMW (n=11). C: Difference in proportion of signal between HMW and SDD (n=11).

[0170] The main objectives of this study were to determine the tissue of origin of the cfDNA present in seminal fluid and to understand whether the source of high molecular weight cfDNA was the same, or different, from that of the small (presumably nucleosomal) cfDNA. To do this, we performed size selection of the cfDNA, followed by enzymatic methylation analysis and tissue deconvolution. FIG. 17A illustrates the tissue deconvolution results of the size selected small DNA (SSD) for 15 men without vasectomies (see Supplemental Figure 1 for results for men with vasectomies). In 13 of the 15 subjects, sperm was by far the most predominant signal contributing to over 70% of the total cfDNA signal. The other 2 subjects had a very high granulocyte signal and less than 20% of the signal was from sperm. The overall predicted prostate fraction for SSD samples was high compared to blood (14), with a median of 2.7% and a max of 20%. Interestingly, the tissue deconvolution results from the size selected high molecular weight (HMW) cfDNA showed an increased proportion of somatic cell signal and decreased proportion of sperm signal (FIG.17B). The proportion of predicted prostate contribution reached as high as 26.5%, with a median of 10.9%, which is of notable significance for the field of liquid biopsy. The HMW DNA showed a marked reduction in sperm signal, with a median reduction of 28.7% compared to matched SSD samples (FIG.17C). Somatic tissues increased in relative predicted fraction in HMW vs SSD samples, with prostate (median +6.2%), granulocyte (median +4.9%), and kidney (median +2.9%) showing predicted increased contribution. These findings demonstrate that the HMW fraction of cfDNA from seminal fluid harbors a higher abundance of somatic cell cfDNA, than the nucleosomal fraction. Supporting Information

[0171] FIG. 18 depicts tissue deconvolution results for vasectomy subjects. A: Tissue Deconvolution Results for SSD. B: Tissue Deconvolution Results for HMW. C: Difference in Proportion of Signal Between HMW and SDD (n=4). It is important to note that the algorithm used in the tissue deconvolution sums the signal to add to 1 (i.e.100%), therefore while it appears that the vasectomy subjects have a lot more signal from the various somatic cells, this is driven by the absence of any sperm signal.

[0172] FIG.19 depicts unsupervised clustering of reference dataset using our deconvolution signature matrix. Pearson correlation followed by clustering of the n=25 methylation markers for each reference tissue show strong agreement within their tissue and mostly poor correlation across tissues. An exception is the slight intermixing of the granulocyte / monocyte marker clusters, which can be explained by their similar developmental origins.

[0173] FIG. 20 depicts values for tissue / cell type specific hypomethylation markers in reference dataset. Each graph represents methylation values for a tissue / cell type specific set of hypomethylationAttorney Ref.60974.00008WO01 (FLW-0006-WO) markers. Shown are methylation values for background (all other tissue / cell types) and the tissue / cell type of interest for all samples in the reference dataset.

[0174] FIG. 21 depicts methylation values for tissue / cell type specific hypomethylation markers in SSD. Each graph represents methylation values for a tissue / cell type specific set of hypomethylation markers. Shown are methylation values for background (reference dataset), tissue of interest (reference dataset), non-vasectomy seminal plasma (SP) samples and vasectomy SP samples. Note that the methylation profile for most of the non-vasectomy SP more closely matches sperm more so than any other tissue type, illustrating sperm to be the most prominent cell type. In vasectomy samples, prostate, granulocytes and monocytes markers appear hypomethylated compared to background tissues, suggesting the presence of DNA from these cell types within these samples.

[0175] FIG. 22 depicts methylation values for tissue / cell type specific hypomethylation markers in HMW. Each graph represents methylation values for a tissue / cell type specific set of hypomethylation markers. Shown are methylation values for background (reference dataset), tissue of interest (reference dataset), non-vasectomy seminal plasma (SP) samples and vasectomy SP samples. Note the very large variability in signal for sperm markers in non-vasectomy samples demonstrating a large range of sperm signal present.

[0176] FIG.23 is a table summarizing the reference datasets used for tissue deconvolution (1-4).

[0177] FIG.24 is a table summarizing the signature matrix. Discussion

[0178] During apoptosis, DNA is fragmented at inter-nucleosomal linker sites and thus the resulting cfDNA is typically around 166 bp, which represents the size of the nucleosome DNA bound to the histone core (146 bp) and a (20 bp) linker connecting the nucleosomes (15). However, cfDNA fragments from tumors and within the fetal fraction of pregnant women tend to be shorter (16). In this study, the electropherograms indicated nucleosomal peaks closer to 200 bp rather than 166 bp, but upon sequencing SSD the mean peak insert size was 146 bp. We believe the insert size to be a more reliable marker of fragment size and believe the larger size seen on electropherogram to be some sort of artifact. Given that 85% of mature sperm DNA is associated with protamines as opposed to histones (17), it also begs the question of whether the sperm signal within the SSD fraction is coming from mature sperm or cells prior to spermiogenesis. Protamine associated DNA forms large toroidal structures of approximately 50,000 kb (18), therefore the majority of cfDNA from apoptotic mature sperm would not be expected to be in the SSD fraction. We also do not expect the HMW fraction to contain an abundance of protamine associated DNA since we deliberately performed a high speed centrifugation step to pellet the protamine toroidal structures. It was interesting to see various unique, but reproducible, cfDNA profiles between subjects. For example, some subjects had a single clean peak of mono- nucleosomal cfDNA, while others had very clear multi-nucleosomal peaks. The underlying biology of this warrants further investigation, but it could be related to the different amounts of DNases present inAttorney Ref.60974.00008WO01 (FLW-0006-WO) semen or the efficiency of cfDNA clearing, which could be of urological clinical significance. Our data also suggests that while increased abstinence times do not change the overall profile of cfDNA (or proportion of small to HWM cfDNA), the total amount of cfDNA increases as a function of abstinence time.

[0179] Previous studies have demonstrated that the yield of cfDNA obtained from seminal fluid is higher than that from other bodily fluids (19), and that fragment size and yield could potentially serve as a prostate cancer biomarker (4,9,10). Ponti, et al (4,9,10) postulated that the high molecular weight DNA seen in prostate cancer patients was derived from necrotic prostate cancer cells (10). However, the exact tissue source of seminal fluid cfDNA has not, until now, been determined. Here we demonstrate for the first time that in men aged 21-60 without vasectomy, most cfDNA usually arises from sperm, but that there is an abundance of cfDNA that comes from various other cell types, including prostate, granulocytes and monocytes. In previous studies, researchers were careful to ensure no sperm lysis during sample collection and processing, and thus claimed that the DNA >1 kb was indeed “cell free” DNA as opposed to an artifact created during collection (19). Another way to rule this out is to look at cfDNA profiles from men who have undergone vasectomy, thus eliminating the chance that sperm is lysed during collection and processing. In this study we observed the absence of a discrete peak of small, nucleosomal cfDNA in participants that had undergone vasectomy, but still a large amount of cfDNA >1 kb. This essentially proves that the larger cfDNA (>1 kb) is coming from cells, at least in part, other than sperm. It is worth noting however, that the majority of this cohort were under 40 yrs of age, and as such, unlikely to have prostate cancer. It is therefore unlikely that in this cohort, the >1 kb cfDNA is coming from necrotic prostate cancer cells.

[0180] Methylation analysis of cfDNA has emerged as a powerful tool for determining the tissue of origin (TOO) of cfDNA fragments, which has significant implications for diagnostics and monitoring of various diseases. To fully understand the TOO of cfDNA in seminal plasma, we performed a series of size selections to purify fragments <500 bp (small cfDNA, SSD) and >800 bp (HMW) and then performed methylation analysis on these two fractions. Our results demonstrate that in men having undergone a vasectomy there was no (significant) sperm signal in either fraction, and that in men not having undergone a vasectomy, the main source of cfDNA in both fractions was usually from sperm. In blood from healthy individuals, more than 90% of cfDNA is derived from white blood cells, with vascular endothelial cells and hepatocytes being the only detectable solid tissue source (14). In this study we demonstrate that prostate derived cfDNA can be highly abundant in healthy controls demonstrating seminal fluid to be a potentially more useful sample type for studying disorders of the prostate. The most striking observation was that the somatic cell signal was usually higher in the HMW fraction than in the SSD fraction. This result is counterintuitive to the world of blood liquid biopsies, whereby the presence of HMW DNA is seen negatively in that it indicates a higher amount of background noise. From a diagnostics perspective, our data suggests that one could improve theAttorney Ref.60974.00008WO01 (FLW-0006-WO) detection sensitivity for various pathological conditions (for example, prostate cancer, prostatitis or benign prostate hyperplasia) by enriching the HMW portion of cfDNA in seminal fluid.

[0181] One limitation of our study is that we did not have access to reference datasets for all tissue types present in the male reproductive tract (for example, testes and epididymis) in building the deconvolution algorithm. Consequently, the proportion estimates of the various tissue / cell types are likely overestimated (given that the algorithm sums the signal to 1). This likely explains why in vasectomy subjects, the level of sperm was not zero. Also, given almost 78% of the testes is thought to consist of spermatogonial stem cells (20), which have almost identical methylation patterns as mature sperm (21), we are likely capturing cfDNA from these cells within the sperm signal. Another limitation is that the cohort of subjects included in this study was limited to males 21-60 yrs of age and limited medical history was obtained. To understand the potential utility of this approach for detection of prostate associated diseases such as cancer, prostatitis or benign prostate hyperplasia, further studies will be required using the relevant patient populations. Example 11 Materials and Methods

[0182] Seminal Fluid Collection: Fresh semen samples were obtained from men aged 21-60 yrs as part of a study approved by an Institutional Review Board. Informed patient consent was obtained for the use of seminal fluid samples and vasectomy status was captured as part of the enrollment. Participants were instructed to collect seminal fluid through masturbation after a period of 2-6 days abstinence. In some cases, participants dropped the sample off at the Fellow laboratory within 2 hours of collection and seminal plasma was then prepared immediately, or preservative added to the seminal fluid sample and left for up to 3 days prior to seminal plasma preparation. In other cases, preservative was added immediately after collection (within 30 minutes) and the sample left for up to 3 days prior to seminal plasma preparation. As part of a stability study, samples were placed in a temperature-controlled chamber and the temperature cycled from 10℃ to 35℃ to mimic shipping conditions.

[0183] Seminal Plasma Preparation: Seminal plasma was prepared by centrifuging the semen at 400 x g for 15 mins to pellet sperm cells and somatic cells. The supernatant was transferred to a new tube and a second spin performed at 16,000 x g for 10 mins to pellet cellular debris and the protamine associated cfDNA from mature sperm.

[0184] cfDNA Extraction and Size Selection: cfDNA was extracted from seminal plasma following Qiagen’s Circulating Nucleic Acids Extraction kit instructions using 1-3 ml seminal plasma. cfDNA was then quantitated using Qubit (Thermo Fisher) and the cfDNA profile obtained from the Tapestation 2200 (Agilent). High molecular weight cfDNA (>800 bp) and small molecular weight cfDNA (<500 bp) were separated as follows: a double size selection was performed using a 0.6x / 1.8x ratio of SPRI reagent to sample volume. The high molecular weight DNA was eluted from the beads and underwent a further size selection using 0.5x ratio of SPRI reagent to sample volume. The high molecular weightAttorney Ref.60974.00008WO01 (FLW-0006-WO) DNA was subject to 250 seconds of sonication using a Covaris ultrasonicator, followed by a SPRI clean up using 1.5x ratio of SPRI reagent to sample volume. After size selection, there was sufficient small sized selected cfDNA (SSD) from 18 subjects (14 non vasectomy and 4 vasectomy) to take into library prep. After size selection, sonication and clean up for the high molecular weight (HMW) DNA, there was sufficient DNA from 15 subjects (11 non vasectomy and 4 vasectomy). 14 subjects (10 non vasectomy and 4 vasectomy) had a paired SSD and HMW sample.

[0185] Methylation Library Preparation and Sequencing: Methylation libraries were prepared using 7- 40 ng of DNA for both SSD and HMW using New England Biolab’s NEBNext® Enyzmatic Methyl- Seq kit. Target enrichment was then performed using Twist’s Human Methylome Panel. Next Generation Sequencing was performed on a NovaSeqX (either on a 10B or 25B flowcell).

[0186] Methylation Computational Processing: Following demultiplexing, reads were trimmed to remove adapters and low quality sequences using fastp (v0.23.4) (22) (extra options: ‘--trim_poly_g -f 1’). Reads were aligned to GRCh38 using bwameth (v0.2.7) (23) and bwa mem2 (v2.2.1) (22). Following alignment, reads were sorted and indexed using samtools (v1.3) (25), and read duplicates were marked using picardtools v3.1.0 (Picard).CpG and CHH methylation content was tabulated using MethylDackel (v0.6.1) (MethylDackel), only within regions covered by the Twist Human Methylome Panel, using the options ‘--CHH --nOT 3,0,0,3 --nOB 0,3,3,0’ to exclude read ends with observed decreased methyl conversion in control materials. For each CpG, the Beta value (methylated reads) / (methylated reads + unmethylated reads) was tabulated.

[0187] Methylation Tissue Deconvolution: Reference tissue datasets were processed from previously published studies (Table S1) (14,26–28). When raw sequencing reads were available, the same methylation processing pipeline described above was used. Otherwise, the processed CpG methylated / unmethylated counts supplied by the study were used to compute Beta values. CpGs were grouped into regions based on the Twist Human Methylome Panel, with the overall methylation Beta value calculated using the median of the CpG level Beta values. For each tissue, 25 marker regions were selected using a one-vs-all approach detailed in Loyfer at al. (14). In short, tissue specific hypomethylated markers were selected based on the difference between the 75th percentile within the given tissue vs the 2.5th percentile for the remaining samples in other tissues. The top 25 markers based on this score were selected, and the tissue signature methylation profile was calculated as the median region level Beta value for all reference samples from the given tissue, restricted to all markers selected across all reference tissues (Table S2).

[0188] A methyl region Beta value was tabulated for each seminal plasma sample, again using the median CpG level Beta value for the given sample. Using the above tissue signature methylation profiles, SciPy non-negative least squares implementation was used to find the optimal coefficients of each tissue contributing to the given sample’s profile. All coefficients were normalized to sum to 1, ensuring estimations could be interpreted as proportions.Attorney Ref.60974.00008WO01 (FLW-0006-WO)

[0189] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. References: 1. Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV; CCGA Consortium. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol.2020 Jun;31(6):745-759. 2. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res.2001 Feb 15;61(4):1659-65. 3. Loyfer, N., Magenheim, J., Peretz, A. et al. A DNA methylation atlas of normal human cell types. Nature 613, 355–364 (2023). 4. Bench GS, Friz AM, Corzett MH, Morse DH, Balhorn R. DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry.1996 Apr 1;23(4):263-71. 5. Brewer LR, Corzett M, Balhorn R. Protamine-induced condensation and decondensation of the same DNA molecule. Science.1999 Oct 1;286(5437):120-3. 6. Moritz L, Hammoud SS. The Art of Packaging the Sperm Genome: Molecular and Structural Basis of the Histone-To-Protamine Exchange. Front Endocrinol (Lausanne).2022 Jun 22;13:895502. 7. Sotolongo B, Lino E, Ward WS. Ability of hamster spermatozoa to digest their own DNA. Biol Reprod.2003 Dec;69(6):2029-35. 8. Samans B, Yang Y, Krebs S, Sarode GV, Blum H, Reichenbach M, Wolf E, Steger K, Dansranjavin T, Schagdarsurengin U. Uniformity of nucleosome preservation pattern in Mammalian sperm and its connection to repetitive DNA elements. Dev Cell.2014 Jul 14;30(1):23-35. 9. Perreault SD, Zirkin BR. Sperm nuclear decondensation in mammals: role of sperm- associated proteinase in vivo. J Exp Zool.1982 Dec 10;224(2):253-7. 10. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod.1996 Oct;55(4):789-95. 11. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev.2016 Sep;35(3):347-76.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 12.Shi J, Zhang R, Li J, Zhang R. Size profile of cell-free DNA: A beacon guiding the practice and innovation of clinical testing. Theranostics.2020 Mar 26;10(11):4737-4748. Liu ZG, Jiao D. Necroptosis, tumor necrosis and tumorigenesis. Cell Stress.2019 Dec 19;4(1):1-8. Ponti G, Maccaferri M, Mandrioli M, Manfredini M, Micali S, Cotugno M, Bianchi G, Ozben T, Pellacani G, Del Prete C, Tomasi A. Seminal Cell-Free DNA Assessment as a Novel Prostate Cancer Biomarker. Pathol Oncol Res.2018 Oct;24(4):941-945. Kim SS, Lee SC, Lim B, Shin SH, Kim MY, Kim SY, Lim H, Charton C, Shin D, Moon HW, Kim J, Park D, Park WY, Lee JY. DNA methylation biomarkers distinguishing early-stage prostate cancer from benign prostatic hyperplasia. Prostate Int.2023 Jun;11(2):113-121. Jamshidi A, Liu MC, Klein EA, Venn O, Hubbell E, Beausang JF, et al. Evaluation of cell-free DNA approaches for multi-cancer early detection. Cancer Cell.2022 Dec 12;40(12):1537- 1549.e12. Odegaard JI, Vincent JJ, Mortimer S, Vowles JV, Ulrich BC, Banks KC, et al. Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies. Clin Cancer Res.2018 Aug 1;24(15):3539–49. Zhu L, Xu R, Yang L, Shi W, Zhang Y, Liu J, et al. Minimal residual disease (MRD) detection in solid tumors using circulating tumor DNA: a systematic review. Front Genet.2023 Aug 10;14:1172108. Ponti G, Maccaferri M, Manfredini M, Micali S, Torricelli F, Milandri R, et al. Quick assessment of cell-free DNA in seminal fluid and fragment size for early non-invasive prostate cancer diagnosis. Clin Chim Acta.2019 Oct;497:76–80. Hu Z, Chen H, Long Y, Li P, Gu Y. The main sources of circulating cell-free DNA: Apoptosis, necrosis and active secretion. Crit Rev Oncol Hematol.2021 Jan;157:103166. Pisareva E, Mihalovičová L, Pastor B, Kudriavtsev A, Mirandola A, Mazard T, et al. Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome- associated circulating DNA. Genome Med.2022 Nov 28;14(1):135. Chou JS, Jacobson JD, Patton WC, King A, Chan PJ. Modified isocratic capillary electrophoresis detection of cell-free DNA in semen. J Assist Reprod Genet.2004 Nov;21(11):397–400. Di Pizio P, Celton N, Menoud PA, Belloc S, Cohen Bacrie M, Belhadri-Mansouri N, et al. Seminal cell-free DNA and sperm characteristic’s: An added biomarker for male infertility investigation. Andrologia.2021 Feb;53(1):e13822. Ponti G, Maccaferri M, Micali S, Manfredini M, Milandri R, Bianchi G, et al. Seminal cell free DNA concentration levels discriminate between prostate cancer and benign prostatic hyperplasia. Anticancer Res.2018 Sep;38(9):5121–5.Attorney Ref.60974.00008WO01 (FLW-0006-WO) Ponti G, Maccaferri M, Mandrioli M, Manfredini M, Micali S, Cotugno M, et al. Seminal Cell-Free DNA Assessment as a Novel Prostate Cancer Biomarker. Pathol Oncol Res.2018 Oct;24(4):941–5. Abramovic I, Pezelj I, Dumbovic L, Skara Abramovic L, Vodopic T, Bulimbasic S, et al. LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA. Prostate.2024 Sep;84(12):1128–37. Medina Diaz I, Nocon A, Mehnert DH, Fredebohm J, Diehl F, Holtrup F. Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing. PLoS ONE.2016 Nov 10;11(11):e0166354. Diaz IM, Nocon A, Held SAE, Kobilay M, Skowasch D, Bronkhorst AJ, et al. Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology. Diagnostics (Basel).2023 Mar 29;13(7). Loyfer N, Magenheim J, Peretz A, Cann G, Bredno J, Klochendler A, et al. A DNA methylation atlas of normal human cell types. Nature.2023 Jan 4;613(7943):355–64. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev.2016 Sep;35(3):347–76. Shi J, Zhang R, Li J, Zhang R. Size profile of cell-free DNA: A beacon guiding the practice and innovation of clinical testing. Theranostics.2020 Mar 26;10(11):4737–48. Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW. Sequence-specific packaging of DNA in human sperm chromatin. Science.1987 May 22;236(4804):962–4. Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol.2007;8(9):227. Li H-G, Huang S-Y, Zhou H, Liao A-H, Xiong C-L. Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non- invasive genetic utilities. Asian J Androl.2009 Nov;11(6):703–9. Islam R, Heyer J, Figura M, Wang X, Nie X, Nathaniel B, et al. T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets. Br J Cancer.2024 Jun;130(12):1893–903. Guo J, Grow EJ, Yi C, Mlcochova H, Maher GJ, Lindskog C, et al. Chromatin and Single- Cell RNA-Seq Profiling Reveal Dynamic Signaling and Metabolic Transitions during Human Spermatogonial Stem Cell Development. Cell Stem Cell.2017 Oct 5;21(4):533-546.e6. Chen S. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta.2023 May 8;2(2):e107. Pedersen BS, Eyring K, De S, Yang IV, Schwartz DA. Fast and accurate alignment of long bisulfite-seq reads. arXiv.2014; Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics.2009 Jul 15;25(14):1754–60.Attorney Ref.60974.00008WO01 (FLW-0006-WO) Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience.2021 Feb 16;10(2). Schrott R, Murphy SK, Modliszewski JL, King DE, Hill B, Itchon-Ramos N, et al. Refraining from use diminishes cannabis-associated epigenetic changes in human sperm. Environ Epigenet.2021 Sep 21;7(1):dvab009. Chen X, Lin Q, Wen J, Lin W, Liang J, Huang H, et al. Whole genome bisulfite sequencing of human spermatozoa reveals differentially methylated patterns from type 2 diabetic patients. J Diabetes Investig.2020 Jul;11(4):856–64. Skvortsova K, Masle-Farquhar E, Luu P-L, Song JZ, Qu W, Zotenko E, et al. DNA hypermethylation encroachment at cpg island borders in cancer is predisposed by H3K4 monomethylation patterns. Cancer Cell.2019 Feb 11;35(2):297-314.e8.

Claims

Attorney Ref.60974.00008WO01 (FLW-0006-WO) CLAIMS 1. A method for enriching a semen sample for a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of nucleosomal exDNA molecules derived from sperm cells.

2. The method of claim 1, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the plurality of nucleosomal exDNA molecules derived from sperm cells.

3. The method of claim 2, wherein the first pellet is generated by centrifuging the semen sample at about 300-500g for about 10-20 minutes.

4. The method of claim 3, wherein the first pellet is generated by centrifuging the sample for 15 minutes.

5. The method of claim 3, wherein the first pellet is generated by centrifuging the sample at 400g.

6. The method of claim 2, wherein the second pellet is generated by centrifuging the isolated first supernatant at about 10,000-18,000g for about 7-15 minutes.

7. The method of claim 6, wherein the second pellet is generated by centrifuging the isolated first supernatant for 10 minutes.

8. The method of claim 6, wherein the second pellet is generated by centrifuging the isolated first supernatant at 16,000g.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 9. The method of claim 1, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine- associated exDNA molecules derived from sperm cells, and wherein the supernatant comprises the plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the supernatant from the pellet, thereby enriching the semen sample for the plurality of nucleosomal exDNA molecules derived from sperm cells.

10. The method of claim 9, wherein the pellet and supernatant are generated by centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes.

11. The method of claim 10, wherein the pellet and the supernatant are generated by centrifuging the sample for 10 minutes.

12. The method of claim 10, wherein the pellet and the supernatant are generated by centrifuging the sample at 16,000g.

13. The method of any one of claims 1-12, further comprising contacting the semen sample with a preservative.

14. The method of claim 13, wherein prior to centrifugation, the semen to preservative ratio ranges from about 0.1 to about 16.

15. The method of any one of claims 1-14, further comprising performing a size-based separation procedure on the DNA fraction.

16. The method of claim 15, wherein performing the size-based separation procedure comprises separating the DNA fraction into a low molecular weight fraction, a high molecular weight fraction, or a combination thereof.

17. The method of claim 16, wherein the low molecular weight fraction comprises exDNA molecules having a size smaller than 500bp, and wherein the high molecular weight fraction comprises exDNA molecules having a size of greater than or equal to 500bp.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 18. The method of claim 17, further comprising performing a size-based separation procedure on the high molecular weight fraction to generate a second low molecular weight fraction comprising exDNA molecules having a size smaller than 800bp, and a second high molecular weight fraction comprising exDNA molecules having a size greater than or equal to 800bp.

19. The method of any one of claims 15-18, wherein the size-based separation procedure comprises a size exclusion chromatography procedure.

20. The method of claim 19, wherein the size exclusion chromatography procedure comprises size selection with solid phase reversible immobilization (SPRI) beads.

21. The method of any one of claims 1-20, which is conducted without freezing the semen sample.

22. A method for enriching a semen sample for a plurality of exDNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of exDNA molecules derived from somatic cells and a plurality of nucleosomal exDNA molecules derived from sperm cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of exDNA molecules derived from somatic cells.

23. The method of claim 22, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the second supernatant from the second pellet, thereby enriching the sample for the plurality of exDNA molecules derived from somatic cells.

24. The method of claim 23, wherein the first pellet is generated by centrifuging the semen sample at about 300-500g for about 10-20 minutes.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 25. The method of claim 24, wherein the first pellet is generated by centrifuging the sample for 15 minutes.

26. The method of claim 24, wherein the first pellet is generated by centrifuging the sample at 400g.

27. The method of claim 23, wherein the second pellet is generated by centrifuging the isolated first supernatant at about 10,000-18,000g for about 7-15 minutes.

28. The method of claim 27, wherein the second pellet is generated by centrifuging the isolated first supernatant for 10 minutes.

29. The method of claim 27, wherein the second pellet is generated by centrifuging the isolated first supernatant at 16,000g.

30. The method of claim 22, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine- associated exDNA molecules derived from sperm cells, and wherein the supernatant comprises the plurality of exDNA molecules derived from somatic cells; and isolating the supernatant from the pellet, thereby enriching the semen sample for the plurality of exDNA molecules derived from somatic cells.

31. The method of claim 30, wherein the pellet and supernatant are generated by centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes.

32. The method of claim 31, wherein the pellet and the supernatant are generated by centrifuging the sample for 10 minutes.

33. The method of claim 31, wherein the pellet and the supernatant are generated by centrifuging the sample at 16,000g.

34. The method of any one of claims 22-33, further comprising contacting the semen sample with a preservative.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 35. The method of claim 34, wherein prior to centrifugation, the semen to preservative ratio ranges from about 0.1 to about 16.

36. The method of any one of claims 22-35, further comprising performing a size-based separation procedure on the DNA fraction.

37. The method of claim 36, wherein performing the size-based separation procedure comprises separating the DNA fraction into a low molecular weight fraction, a high molecular weight fraction, or a combination thereof.

38. The method of claim 37, wherein the low molecular weight fraction comprises exDNA molecules having a size smaller than 500bp, and wherein the high molecular weight fraction comprises exDNA molecules having a size of greater than or equal to 500bp.

39. The method of claim 38, further comprising performing a size-based separation procedure on the high molecular weight fraction to generate a second low molecular weight fraction comprising exDNA molecules having a size smaller than 800bp, and a second high molecular weight fraction comprising exDNA molecules having a size greater than or equal to 800bp.

40. The method of any one of claims 36-39, wherein the size-based separation procedure comprises a size exclusion chromatography procedure.

41. The method of claim 40, wherein the size exclusion chromatography procedure comprises size selection with solid phase reversible immobilization (SPRI) beads.

42. The method of any one of claims 22-41, which is conducted without freezing the semen sample.

43. The method of any one of claims 22-42, further comprising analyzing the plurality of exDNA molecules derived from sperm cells in the semen sample.

44. The method of any one of claims 22-43, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules secreted by somatic cells in the semen sample.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 45. The method of any one of claims 22-43, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample.

46. The method of claim 45, wherein somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

47. The method of any one of claims 22-46, wherein somatic cells in the semen sample comprise prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof.

48. A method for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules from the somatic cells; centrifuging the semen sample to generate a DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells; and isolating the DNA fraction, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

49. The method of claim 48, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a first pellet and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

50. The method of claim 49, wherein the first pellet and the first supernatant are generated by centrifuging the semen sample at about 300-500g for about 10-20 minutes.

51. The method of claim 50, wherein the first pellet and the first supernatant are generated by centrifuging the sample for 15 minutes.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 52. The method of claim 50, wherein the first pellet and the first supernatant are generated by centrifuging the sample at 400g.

53. The method of claim 49, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant at about 10,000-18,000g for about 7-15 minutes.

54. The method of claim 53, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant for 10 minutes.

55. The method of claim 53, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant at 16,000g.

56. The method of claim 49, wherein the first pellet comprises a plurality of intact sperm cells, a plurality of intact somatic cells, or any combination thereof.

57. The method of claim 49, wherein the second pellet comprises a plurality of intact sperm cells, a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine- associated exDNA molecules derived from sperm cells, or any combination thereof.

58. The method of claim 48, wherein centrifuging the semen sample to generate the DNA fraction comprises: centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and wherein the supernatant comprises the plurality of DNA molecules derived from the somatic cells; and isolating the supernatant from the pellet, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

59. The method of claim 58, wherein the pellet and supernatant are generated by centrifuging the semen sample at about 10,000-18,000g for about 7-15 minutes.

60. The method of claim 59, wherein the pellet and the supernatant are generated by centrifuging the sample for 10 minutes.

61. The method of claim 59, wherein the pellet and the supernatant are generated by centrifuging the sample at 16,000g.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 62. The method of claim 58, wherein the pellet comprises a plurality of intact sperm cells, a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, or any combination thereof.

63. The method of claim 57 or claim 62, wherein cellular debris from sperm cells that have undergone natural cell lysis comprises cellular debris from sperm cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

64. The method of claim57 or claim 62, wherein the cellular debris from lysed somatic cells comprises a plurality of somatic cells in the semen sample that have undergone natural cell lysis, a plurality of somatic cells in the semen sample selectively lysed by the lysis reagent, or any combination thereof.

65. The method of claim 64, wherein the plurality of somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

66. The method of any one of claims 48-65, further comprising contacting the semen sample with a preservative.

67. The method of claim 66, wherein prior to centrifugation, the semen to preservative ratio ranges from about 0.1 to about 16.

68. The method of any one of claims 48-67, wherein the somatic cells comprise prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof.

69. The method of any one of claims 48-68, which is conducted without freezing the semen sample.

70. The method of any one of claims 48-68, wherein the DNA fraction comprises a plurality of exDNA molecules derived from sperm cells in the semen sample.

71. The method of any one of claims 48-68, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 72. The method of claim 71, wherein the plurality of exDNA molecules derived from somatic cells in the semen sample comprises a plurality of exDNA molecules secreted by somatic cells in the semen sample.

73. The method of claim 71, wherein the plurality of exDNA molecules derived from somatic cells in the semen sample comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample.

74. The method of claim 73, wherein somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

75. A method for enriching a semen sample for a plurality of DNA molecules derived from somatic cells in the semen sample, the method comprising: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules from the somatic cells and the somatic cell lysis reagent; centrifuging the mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, thereby enriching the semen sample for the plurality of DNA molecules derived from somatic cells.

76. The method of claim 75, further comprising: isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; and isolating the third supernatant from the third pellet, thereby enriching the sample for the plurality of exDNA molecules derived from somatic cells in the semen sample.

77. The method of claim 76, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules secreted by somatic cells in the semen sample.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 78. The method of claim 76, wherein the plurality of exDNA molecules derived from somatic cells in the semen comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample.

79. The method of claim 78, wherein somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

80. The method of claim 75 or 76, wherein the first pellet and the first supernatant are generated by centrifuging the semen sample at about 300-500g for about 10-20 minutes.

81. The method of claim 80, wherein the first pellet and the first supernatant are generated by centrifuging the sample for 15 minutes.

82. The method of claim 80, wherein the first pellet and the first supernatant are generated by centrifuging the sample at 400g.

83. The method of claim 75, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant at about 10,000-18,000g for about 7-15 minutes.

84. The method of claim 83, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant for 10 minutes.

85. The method of claim 83, wherein the second pellet and the second supernatant are generated by centrifuging the isolated first supernatant at 16,000g.

86. The method of claim 76, wherein the third pellet and the third supernatant are generated by centrifuging the isolated first supernatant at about 10,000-18,000g for about 7-15 minutes.

87. The method of claim 86, wherein the third pellet and the third supernatant are generated by centrifuging the isolated first supernatant for 10 minutes.

88. The method of claim 86, wherein the third pellet and the third supernatant are generated by centrifuging the isolated first supernatant at 16,000g.

89. The method of claim 75 or claim 76, wherein the first pellet comprises a plurality of intact sperm cells, a plurality of intact somatic cells, or any combination thereof.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 90. The method of claim 75, wherein the second pellet comprises a plurality of unlysed somatic cells, cellular debris from lysed somatic cells, cellular debris from sperm cells that have undergone natural cell lysis, a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, or any combination thereof.

91. The method of claim 90, wherein cellular debris from sperm cells that have undergone natural cell lysis comprises cellular debris from sperm cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

92. The method of claim 90, wherein cellular debris from lysed somatic cells comprises a plurality of somatic cells in the semen sample that have undergone natural cell lysis, a plurality of somatic cells in the semen sample selectively lysed by the lysis reagent, or any combination thereof.

93. The method of claim 92, wherein the plurality of somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

94. The method of any one of claims 75-93, further comprising contacting the semen sample with a preservative.

95. The method of claim 94, wherein prior to centrifugation, the semen to preservative ratio ranges from about 0.1 to about 16.

96. The method of any one of claims 75-95, wherein the somatic cells comprise prostate cells, testicular cells, kidney cells, bladder cells, white blood cells, or any combination thereof.

97. The method of any one of claims 75-96, which is conducted without freezing the semen sample.

98. The method of any one of claims 75-97, further comprising analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

99. A method for analyzing a quantity of somatic cell derived DNA molecules from a specific somatic cell type in a semen sample, the method comprising:Attorney Ref.60974.00008WO01 (FLW-0006-WO) isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the DNA fraction to determine an abundance of somatic cell derived DNA molecules from the specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a sufficient quantity of the somatic cell derived DNA molecules from the specific somatic cell type for analysis of a prostate- specific disorder.

100. The method of claim 99, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

101. The method of claim 99, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a pellet and a supernatant; and isolating the supernatant from the pellet, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, wherein the supernatant comprises the DNA fraction, and wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

102. The method of claim 99, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the plurality of somatic cells; centrifuging the semen sample to generate a first pellet and a first supernatant;Attorney Ref.60974.00008WO01 (FLW-0006-WO) isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from the plurality of somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

103. The method of claim 99, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules derived from the somatic cells; centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells; and isolating the supernatant from the pellet, wherein the supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules from the somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

104. The method of claim 99, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules derived from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules derived from the somatic cells and the somatic cell lysis reagent; centrifuging the mixture to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells.

105. The method of claim 104, further comprising: isolating the first supernatant from the first pellet;Attorney Ref.60974.00008WO01 (FLW-0006-WO) centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; and isolating the third supernatant from the third pellet, wherein the third supernatant comprises a second DNA fraction that comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells.

106. The method of claims 99, 100, 101, 102, 103, or 105, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample.

107. The method of claim 106, wherein the somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

108. The method of any one of claims 99-106, further comprising analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

109. The method of claims 99, 100, 101, 102, 103, or 105, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules secreted by somatic cells in the semen sample.

110. The method of any one of claims 99-106, wherein the specific somatic cell type and / or the plurality of somatic cells comprise prostate cells, prostate cancer cells, white blood cells, testicular cells, kidney cells, bladder cells, or any combination thereof.

111. The method of any one of claims 99-106, wherein the prostate-specific disorder is prostate cancer or testicular cancer.

112. The method of any one of claims 99-106, wherein the prostate-specific disorder is benign prostatic hyperplasia (BPH).

113. The method of any one of claims 99-106, wherein the prostate-specific disorder is prostatitis.

114. The method of any one of claims 99-106, further comprising performing a size-based separation procedure on the DNA fraction.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 115. The method of claim 114, wherein performing the size-based separation procedure comprises separating the DNA fraction into a low molecular weight fraction, a high molecular weight fraction, or a combination thereof.

116. The method of claim 115, wherein the low molecular weight fraction comprises somatic cell derived DNA molecules having a size smaller than 500bp, and wherein the high molecular weight fraction comprises somatic cell derived DNA molecules having a size of greater than or equal to 500bp.

117. The method of claim 116, further comprising performing a size-based separation procedure on the high molecular weight fraction to generate a second low molecular weight fraction comprising somatic cell derived DNA molecules having a size smaller than 800bp, and a second high molecular weight fraction comprising somatic cell derived DNA molecules having a size greater than or equal to 800bp.

118. The method of any one of claims 114-116, wherein the size-based separation procedure comprises a size exclusion chromatography procedure.

119. The method of claim 118, wherein the size exclusion chromatography procedure comprises size selection with solid phase reversible immobilization (SPRI) beads.

120. The method of any one of claims 99-119, wherein comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a sufficient quantity of the somatic cell derived DNA molecules from the specific somatic cell type for analysis of a prostate-specific disorder comprises performing a tissue of origin analysis.

121. The method of any one of claim 99-120, further comprising contacting the semen sample with a preservative.

122. The method of any one of claims 99-121, which is conducted without freezing the semen sample.

123. A diagnostic method for detecting a prostate-specific disorder from a semen sample, the method comprising:Attorney Ref.60974.00008WO01 (FLW-0006-WO) isolating a DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample; performing a sequencing analysis on the plurality of somatic cell derived DNA molecules to determine an abundance of somatic cell derived DNA molecules from a specific somatic cell type in the semen sample; and comparing the abundance of somatic cell derived DNA molecules derived from cells of the specific somatic cell type to a threshold value to determine whether the semen sample contains a clinically relevant prostate-specific disorder signal.

124. The diagnostic method of claim 123, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first pellet comprising a plurality of intact sperm cells and a plurality of intact somatic cells, and a first supernatant; isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a second pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

125. The diagnostic method of claim 123, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a pellet and a supernatant; and isolating the supernatant from the pellet, wherein the pellet comprises a plurality of intact sperm cells and a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells, and wherein the supernatant comprises the DNA fraction, wherein the DNA fraction comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

126. The diagnostic method of claim 123, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules from the somatic cells; centrifuging the semen sample to generate a first pellet and a first supernatant; isolating the first supernatant from the first pellet;Attorney Ref.60974.00008WO01 (FLW-0006-WO) centrifuging the isolated first supernatant to generate a second pellet and a second supernatant; and isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from the plurality of somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

127. The diagnostic method of claim 123, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: contacting the semen sample with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the semen sample and release a plurality of DNA molecules from the somatic cells; centrifuging the semen sample to generate a pellet and a supernatant, wherein the pellet comprises a plurality of high molecular weight protamine-associated exDNA molecules derived from sperm cells; and isolating the supernatant from the pellet, wherein the supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules from the somatic cells, a plurality of exDNA molecules derived from somatic cells in the semen sample, and a plurality of nucleosomal exDNA molecules derived from sperm cells in the semen sample.

128. The diagnostic method of claim 123, wherein isolating the DNA fraction comprising a plurality of somatic cell derived DNA molecules of the semen sample comprises: centrifuging the semen sample to generate a first cell pellet and a first supernatant; treating the first cell pellet with a somatic cell lysis reagent to selectively lyse a plurality of somatic cells in the first cell pellet and release a plurality of DNA molecules from the somatic cells into the somatic cell lysis reagent to generate a mixture comprising the plurality of DNA molecules from the somatic cells and the somatic cell lysis reagent; and centrifuging the mixture to generate a second pellet and a second supernatant; isolating the second supernatant from the second pellet, wherein the second supernatant comprises the DNA fraction, wherein the DNA fraction comprises the plurality of DNA molecules derived from somatic cells.

129. The diagnostic method of claim 128, further comprising: isolating the first supernatant from the first pellet; centrifuging the isolated first supernatant to generate a third pellet comprising a plurality of cellular debris and high molecular weight protamine-associated exDNA molecules derived from sperm cells and a third supernatant; andAttorney Ref.60974.00008WO01 (FLW-0006-WO) isolating the third supernatant from the third pellet, wherein the third supernatant comprises a second DNA fraction that comprises a plurality of exDNA molecules derived from somatic cells in the semen sample and a plurality of nucleosomal exDNA molecules derived from sperm cells.

130. The diagnostic method of claims 123, 124, 125, 126, 127, or 129, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules derived from somatic cells that have undergone natural cell lysis in the semen sample.

131. The diagnostic method of claim 130, wherein the somatic cells in the semen sample that have undergone natural cell lysis comprise somatic cells that have undergone necrosis, apoptosis, NETosis, or some other form of cell lysis during collection, transportation, or storage of the semen sample.

132. The diagnostic method of any one of claims 123-130, further comprising analyzing a plurality of exDNA molecules derived from sperm cells in the semen sample.

133. The diagnostic method of claims 123, 124, 125, 126, 127, or 129, wherein the plurality of exDNA molecules derived from somatic cells comprises a plurality of exDNA molecules secreted by somatic cells in the semen sample.

134. The diagnostic method of any one of claims 123-130, wherein the specific somatic cell type and / or the plurality of somatic cells comprise prostate cells, prostate cancer cells, white blood cells, testicular cells, kidney cells, bladder cells, or any combination thereof.

135. The diagnostic method of any one of claims 123-130, wherein the prostate-specific disorder is prostate cancer or testicular cancer.

136. The diagnostic method of any one of claims 123-130, wherein the prostate-specific disorder is benign prostatic hyperplasia (BPH).

137. The diagnostic method of any one of claims 123-130, wherein the prostate-specific disorder is prostatitis.

138. The diagnostic method of any one of claims 123-130, further comprising performing a size- based separation procedure on the DNA fraction.Attorney Ref.60974.00008WO01 (FLW-0006-WO) 139. The diagnostic method of claim 138, wherein performing the size-based separation procedure comprises separating the DNA fraction into a low molecular weight fraction, a high molecular weight fraction, or a combination thereof.

140. The diagnostic method of claim 139, wherein the low molecular weight fraction comprises somatic cell derived DNA molecules having a size smaller than 500bp, and wherein the high molecular weight fraction comprises somatic cell derived DNA molecules having a size of greater than or equal to 500bp.

141. The diagnostic method of claim 140, further comprising performing a size-based separation procedure on the high molecular weight fraction to generate a second low molecular weight fraction comprising somatic cell derived DNA molecules having a size smaller than 800bp, and a second high molecular weight fraction comprising somatic cell derived DNA molecules having a size greater than or equal to 800bp.

142. The diagnostic method of any one of claims 138-141, wherein the size-based separation procedure comprises a size exclusion chromatography procedure.

143. The diagnostic method of claim 142, wherein the size exclusion chromatography procedure comprises size selection with solid phase reversible immobilization (SPRI) beads.

144. The diagnostic method of any one of claims 123-143, wherein comparing the abundance of somatic cell derived DNA molecules from the specific somatic cell type to a threshold value to determine whether the semen sample contains a clinically relevant prostate-specific disorder signal comprises performing a tissue of origin analysis.

145. The diagnostic method of any one of claims 123-144, further comprising contacting the semen sample with a preservative.

146. The diagnostic method of any one of claims 123-145, which is conducted without freezing the semen sample.

147. The method of any one of claims 2, 23, 49, 100, 102, 124, and 126, wherein the first pellet is generated by centrifuging the semen sample at about 16,000g, and wherein the second pellet is generated by centrifuging the semen sample at about 3,000-10,000g.

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