US 20110201047Al (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2011/0201047 A1 Paduch (43) Pub. Date: Aug. 18, 2011

(54) NOVEL SORTING TECHNOLOGY THAT (2), (4) Date: Apr. 14, 2011 ALLOWS FOR HIGHLY EFFICIENT . . SELECTION OF SPERM WITHOUT Related US. Application Data CHROMATIN DAMAGE (60) Provisional application No. 61/ 105,223, ?led on Oct. 14, 2008. (75) Inventor: Darius A. Paduch, Jersey City, NJ PllblicatiOIl Classi?catiOIl (Us) (51) Int. Cl. C12Q 1/02 (2006.01) (73) Assignee; CORNELL UNIVERSITY, Ithaca, (52) US. Cl...... 435/29 NY (Us) (57) ABSTRACT This invention provides a method to select and enrich healthy (21) APP1~ N05 13/124,246 sperm for use in assisted reproductive techniques comprising incubating sperm With at least one marking reagent that enters 22 PCT F-I d, 0 L 14 2009 unhealthy sperm but not healthy sperm. The invention also ( ) 1 e c ’ provides a method to sort healthy sperm of high viability from apoptotic, necrotic and dead sperm by using YO-PRO-l and (86) PCT No.: PCT/US09/60636 PI for ?uorescence activated cell sorting in How cytometers. A kit comprising YO-PRO-l and PI is also included in this § 371 (c)(1), invention.

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NOVEL SORTING TECHNOLOGY THAT have absorbed the dye are identi?able by the ?uorescence of ALLOWS FOR HIGHLY EFFICIENT the dye and can be separated by knoWn cell-sorting proce SELECTION OF SPERM WITHOUT dures. When YO-PRO-l is used in conjunction With pro CHROMATIN DAMAGE pidium iodide, a Well knoWn ?uorescent marker of dead cells, both apoptotic and dead cells can be identi?ed and removed BACKGROUND from the sperm sample prior to its use in assisted reproductive [0001] Most in vitro fertilization (IVF) failures result from techniques, improving the possibility of successful fertiliza male factor de?ciencies. The quality of sperm is one of the tion, implantation, gestation, and birth of healthy offspring. factors determining the success rate of IVF. The use of ran [0005] The method of the present invention permits reduc domly selected sperm for this procedure can result in various tion of unhealthy sperms in a semen sample, hence enrich anomalies of sperm decondensation and embryonic develop ment of healthy sperms in the same sample. By “reduction of ment. Currently, sperm quality is evaluated by conventional unhealthy sperms”, it is meant that the number of unhealthy semen analysis using a light microscope to determine sperm sperms in a sperm sample or population is reduced by at least concentration, motility and morphology. However, conven tWo (2) fold, at least 3 fold, at least 3 fold, at least 5 fold, at tional semen analysis has limited clinical value for predicting least 6 fold, or at least 7 fold or more. the success rate of IVF as 50% of couples With failed fertili [0006] The advantage of this approach is that the integrity zation have normal pre-IVF semen analysis. Furthermore, of healthy sperms in the sample is preserved, so that the conventional semen analysis does not assess the presence of remaining healthy sperm cells can still be used in assisted apoptotic spermatozoa, Which might be partially responsible reproductive techniques. for the loW fertilization and implantation rates in assisted [0007] The method may be used With human sperms, or reproduction. Sperm chromatin damage may be a negative With sperms from animals such as bulls, stallions, and dogs. predictor for IVF outcomes in couples With recurrent spon taneous abortions and poor embryo development. Methods DEFINITIONS for detecting chromatin damage such as SCSA, TUNEL, Healthy Sperm Halosperrn and Comet assays require permanent ?xation of sperm, making the them unusable for in vitro fertilization or [0008] Healthy sperms refers to sperms that have normal agricultural insemination for livestock and pets. Early apop morphology and motility, are not apoptotic, necrotic, or dead, totic events resulting in chromatin damage are usually accom and are able to produce a fertilized egg naturally or When panied by increased permeability of the cell membrane to introduced to an egg using assisted reproductive techniques. large ions. In this invention, the use of marking reagent is disclosed for ?uorescence activated cell sorting to identify Apoptosis apoptotic sperm With chromatin damage, and sort out healthy [0009] Apoptosis refers broadly to any “programmed cell sperm for use in assisted reproductive technology. death” in Which single or groups of cells that are part of a multicellular organism die in a regulated process. Broadly SUMMARY speaking necrosis is the other form of cell death in a multi [0002] This invention provides a method to select and cellular organism, and is the process by Which cells die under enrich healthy sperms for use in assisted reproduction tech stress, disease, or other attack. niques comprising: a) incubating sperms With at least one [0010] The characteristic morphology of apoptotic cells marking reagent that enters unhealthy sperm cells but not includes increases in the permeability of the plasma mem healthy sperm cells; b) sorting marked sperm cells from brane to Water and large ions, loss of membrane asymmetry unmarked sperm cells. The unhealthy cells can be apoptotic, and attachment, formation of irregular bulges in the cell mem necrotic, or dead. The marking reagents that may be used here brane caused by localized decoupling of the cytoskeleton include ?uorescent dyes such as YO-PRO-l and propidium from the plasma membrane, cell shrinkage, nuclear fragmen iodide (PI). A ?oW cytometer can be used to sort marked tation, chromatin condensation, and chromosomal DNA frag sperm from unmarked sperm. A kit is provided, comprising mentation. Apoptosis is different from necrosis, as the pro YO-PRO-l and propidium iodide (PI). cesses associated With apoptosis in disposal of cellular debris do not damage the organism. BRIEF DESCRIPTION OF THE FIGURES Apoptotic Sperm [0003] FIG. 1: Semen samples from 18 men Were analyzed With propidium iodide (PI) and YO-PRO-l. Dead sperm are [0011] Apoptotic sperms are sperms that are going through permeable to PI, and apoptotic sperm are permeable to YO apopto sis. An early apoptotic event in cells is characterized by PRO-l. Intact cells are impermeable to both. Hoechst 33342 increases in the permeability of plasma membranes and loss Was used to calibrate the BD LSR II ?oW cytometer. Semen of phospholipid asymmetry. Since successful fertilization samples from ?ve patients are sorted With the BD FACS requires a sperm plasma membrane With normal integrity and Vantage. Healthy sperm Were separated from necrotic and function, apoptotic sperm Will have very loW fertility. apoptotic cells based on ?uorochrome staining. Necrosis DETAILED DESCRIPTION [0012] Necrosis refers to the unnatural death of cells and [0004] This invention involves the use of marking reagents living tissue. It begins With cell sWelling, chromatin diges to label unhealthy sperm cells. In particular, apoptotic cells tion, and disruption of the plasma membrane and organelle are permeable by YO-PRO-l, While healthy sperms are not. membranes. Late necrosis is characterized by extensive DNA Necrotic and dead sperms are permeable by propidium iodide hydrolysis, vacuolation of the endoplasmic reticulum, and healthy sperms are not. The unhealthy sperm cells that organelle breakdoWn, and cell lysis. In contrast to apoptosis, US 2011/0201047 A1 Aug. 18,2011

cleanup of cell debris by phagocytes of the immune system is [0030] Gestational Carrier generally more dif?cult, as the disorderly death generally [0031] A gestational carrier is an option When a patient’s does not send signals to nearby phagocytes to engulf the medical condition prevents a safe pregnancy, When a patient dying cell. This lack of signaling makes it harder for the has ovaries but no uterus due to congenital absence or previ immune system to locate and recycle dead cells Which have ous surgical removal, and Where a patient has no ovaries and died through necrosis than if the cell had undergone apopto is also unable to carry a pregnancy to full term. sis. [0032] Cryopreservation [0033] Eggs, sperm and reproductive tissue can be pre Assisted Reproductive Techniques served for later IVF. [0013] Assisted reproductive techniques (ART) is a general [0034] The folloWing Assisted Reproduction techniques term referring to methods used to achieve pregnancy by arti don’t necessarily involve IVF. ?cial or partially arti?cial means. In general, ARTs involve [0035] Gamete intrafallopian transfer (GIFT) surgically removing eggs from a female, obtaining sperm [0036] In gamete intrafallopian transfer a mixture of sperm from a male, combining the egg With sperm in the laboratory, and eggs is placed directly into a Woman’s fallopian tubes and at some point returning the fertilized egg or embryo to a using laparoscopy folloWing a transvaginal ovum retrieval. female body for gestation. ART also include treatments in [0037] Preimplantation Genetic Diagnosis (PGD) Which only sperm are handled (i.e., arti?cial insemination) or [0038] PGD involves the use of Fluorescent In Situ Hybrid procedures in Which a Woman takes medicine only to stimu ization (FISH) or Polymerase Chain Reaction (PCR) DNA late egg production Without the intention of having eggs ampli?cation to help identify genetically abnormal embryos retrieved. and improve healthy outcomes. [0014] When ARTs are used in non-human animals, the [0039] Sex Selection process is generally called arti?cial insemination. [0040] Sex selection is the attempt to control the sex of [0015] Examples of ARTs include, but are not limited to: offspring to achieve a desired sex. It can be accomplished in [0016] In vitro Fertilization several Ways, both pre- and post-implantation of an embryo, [0017] In vitro fertilization (IVF) is the technique of letting as Well as at birth. Pre-implantation techniques include POD, fertilization of the male and female gametes (sperm and egg) but also sperm sorting. occur outside the female body. [0041] Arti?cial Insemination [0018] Transvaginal Ovum Retrieval (OCR) [0042] Arti?cial insemination (AI) is When sperm is placed [0019] This is the process Whereby a small needle is into a female’s uterus (intrauterine) or cervix (intracervical) inserted through the back of the vagina and guided via ultra using arti?cial means rather than by natural copulation. sound into the ovarian follicles to collect the ?uid that con [0043] Therapeutic Donor Insemination tains the eggs. [0044] Therapeutic donor is an expansion of arti?cial [0020] Assisted Zona Hatching (AZH) insemination. It is also called arti?cial insemination by donor [0021] Assisted zona hatching is performed shortly before and is used in situations Where the Woman doesn’t have a the embryo is transferred to the uterus. A small opening is partner With functional sperm. Instead, a sperm donor sup made in the outer layer surrounding the egg in order to help plies the sperm. the embryo hatch out and aid in the implantation process of [0045] Surgical Sperm Retrieval (SSR) the groWing embryo. [0046] The reproductive urologist may obtain sperm from [0022] Intracytoplasmic Sperm Injection (ICSI) the vas deferens, epididymis or directly from the testis in a [0023] Intracytoplasmic sperm injection is bene?cial in the short outpatient procedure. case of male factor infertility Where sperm counts are very [0047] Frozen Embryo Transfer (FET) loW or failed fertilization occurred With previous IVF attempt [0048] A fertilized embryo can be cryopreserved. The latter (s). The ICSI procedure involves a single sperm carefully insertion in the body is by the technique Frozen Embryo injected into the center of an egg using a microneedle. Transfer (FET). [0024] Autologous Endometrial Coculture [0049] Marking Reagents [0025] Autologous endometrial coculture is a possible [0050] The term marking reagent refers to any kind of label treatment for patients Who have failed previous IVF attempts or reagent that can give a readable signal indicating the exist or Who have poor embryo quality. The patient’s fertilized eggs ence of a molecule, the presence (past or present) of living are placed on top of a layer of cells from the patient’s oWn organisms, the physiological state of living cells, disease uterine lining, creating a more natural environment for conditions, viral infections or a physical process. embryo development. [0051] For example, marking reagents may also be used to [0026] Zygote Intrafallopian Transfer (ZIFT) mark cells that are dead or undergoing necrosis. [0027] In zygote intrafallopian transfer egg cells are [0052] Reagents that mark cells that are dead or undergoing removed from the Woman’s ovaries and fertilized in the labo necrosis include, but are not limited to: propidium iodide. ratory; the resulting zygote is then placed into the fallopian [0053] For example, marking reagents may be used to mark tube. cells that are undergoing apoptosis. [0028] Egg Donor [0054] Marking reagents for apoptotic cells include, but are [0029] Egg donors are resources for Women With no eggs not limited to: due to surgery, chemotherapy, or genetic causes; or With poor [0055] Reagents that only enter and stain cells that are egg quality, previously unsuccessful IVF cycles or advanced apoptotic, and Will not enter healthy cells. For example, YO maternal age. In the egg donor process, eggs are retrieved PRO-1. from a donor’s ovaries, fertilized in the laboratory With the [0056] Reagent that mainly enters cells that apoptotic and sperm from the recipient’s partner, and the resulting healthy shoWs strong staining, but only enters healthy cells in minor embryos are returned to the recipient’s uterus. amounts and stains them dimly. For example, Hoechst 33342. US 2011/0201047 A1 Aug. 18,2011

[0057] Reagents that Will only bind to certain cell surface difference Was highly statistically signi?cant (p<0.00001). molecules of apoptotic cells, but not healthy cells. Unstained sperm had excellent progressive motility and nor [0058] Reagent that Will only bind to certain cell surface mal morphology. molecules of apoptotic cells include, but are not limited to: [0065] This technology alloWs for sperm chromatin dam Annexin V, Which bind to phosphatidylserine that are redis age analysis as Well as quick and reliable sorting, separating tributed from the inner plasma membrane lea?et to the out normal sperm from those With chromatin damage. Because lea?et during the early onset of apopotisis. When. AnnexinV the test employs large molecules that require activation of is conjugated With ?uorescent chemicals or magnetic beads, it sodium channels to enter sperm, there is little risk for residual can give out readable signals for identi?cation of apoptotic ?uorochrome in the isolated specimen. This assay may be a cells. neW treatment modality for couples With male factor infertil [0059] Reagents that bind to overexpressed key proteins of ity secondary to sperm chromatin damage. apoptosis-related pathWays. Reagents that bind to overex [0066] The folloWing statements are potential claims that pressed key proteins of apoptosis-related pathWays include, may be converted to claims in a future application. No modi but are not limited to: antibodies against Cytochrome c, ?cations of the folloWing statements should be alloWed to cleaved Caspase-3, cleaved PARP (poly-ADP-ribose-poly affect the interpretation of claims Which may be drafted When merase), Fas, Bcl-x, and p53. When these antibodies are this provisional application is converted into a regular utility conjugated With ?uorescent chemicals or magnetic beads, application. they can give out readable signals for identi?cation of apop 1. A method to select healthy sperm for use in assisted totic cells. reproductive technologies comprising: a) incubating sperm With at least one marking reagent that Methods to Separate Cells enters unhealthy sperm cells but not healthy sperm cells, [0060] Several methods to separate cells that are marked and b) separating marked sperm from unmarked sperm. 2. A method according to claim 1, Wherein the unhealthy from cells that are unmarked are knoWn in the art. The term “marked cells” refers to cells Which a marking reagent has sperm are apoptotic. 3. A. method according to claim 1 Wherein the unhealthy bound to and in some instances penetrated into. [0061] Separation methods include, but are not limited to: sperm are necrotic. [0062] Fluorescence-activated cell sorting (FACS), Mag 4. A method according to claim 1, Wherein the reagent netic-activated cell sorting (MACS), density gradient cell ?uoresces. sorting, selection of desired cells by sedimentation, a?inity 5. A method according to claim 1, Wherein the ?uorescent adsorption or a?inity extraction. reagent is YO-PRO-l. 6. A method according to claim 1, Wherein the ?uorescent EXAMPLES reagent is propidium iodide. 7. A method according to claim 1, Wherein sperms are [0063] Semen samples from 18 men Were analyZed using incubated With bothYO-PRO-l and propidium iodide prior to propidium iodide (PI) andYO-PRO-l (PF-1). Dead sperm are the separating step. permeable to PI, and apoptotic cells are permeable to PF-l. 8. A method according to claim 1, Wherein the sperm is Intact cells are impermeable to both. Hoechst 33342 Was used sorted using a ?oW cytometer. to calibrate the BD LSR II ?oW cytometer. Semen samples 9. A method of reducing unhealthy sperms in a semen from ?ve patients Were sorted With the BD FACSVantage. sample for use in assisted reproductive technologies compris Normal spermatozoa Were separated from necrotic and apo mg: ptotie cells based on ?uorochrome staining. Percoll density a) incubating said semen sample With at least one marking gradient Was used to remove debris prior to sorting. To verify reagent that enters unhealthy sperm cells but not healthy selection of intact sperm, the stained and unstained popula sperm cells in said sample, and tions Were examined microscopically for motility and viabil b) separating marked sperms from unmarked sperm, ity, and analyZed for DNA fragmentation With the TUNEL thereby reducing unhealthy sperms in said sample. assay. The tWo populations Were compared using the chi 10. The method of claim 9, Wherein the amount of square test for difference in the percentage of TUNEL-posi unhealthy sperms in said sample is reduced by at least tWo to tive cells. seven fold. [0064] RESULTS: In the group positive for PF-l and Pl, 11. A kit comprising YO-PRO-l and propidium iodide 431 of 2,167 sperm (19.5%) Were TUNEL positive. In the non-staining group only 33 of 2,263 (1.5%) ?uoresced. The * * * * *