Claims:I/WE CLAIM:
1) A method for analysing azoospermia, wherein the method comprises,
collecting a testicular tissue from a testis of a subject/human using a minimally invasive method;
performing a deoxyribonucleic acid (DNA) flow cytometric analysis on the collected testicular tissue;
identifying a chromosomal status of spermatogenesis in the testis based on the result of DNA flow cytometric analysis; and
determining etiology of the azoospermia in the patient based on the status of the spermatogenesis.
2) The method as claimed in claim 1, wherein the minimally invasive method comprises obtaining the testicular tissue using a fine needle aspiration cytology (FNAC) method.
3) The method as claimed in claim 1, comprising isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis.
4) The method as claimed in claim 3, wherein performing the DNA flow cytometric analysis comprises isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis with a nucleic acid binding dye and analysing amount of dye binding and fluorescence intensity of the dye.
5) The method as claimed in claim 4, wherein the nucleic acid binding dye comprises propidium iodide.
6) The method as claimed in claim 1, wherein identifying the chromosomal status of spermatogenesis comprises identification of presence or populations of haploid cells, diploid cells, tetraploid cells, or combinations thereof.
7) The method as claimed in claim 6, wherein determining etiology of the azoospermia in the patient comprises identifying obstructive azoospermia, meiotic arrest, or premeiotic arrest, based on the presence or populations of the haploid cells, diploid cells, tetraploid cells, or combinations thereof in the testicular tissue.
8) The method as claimed in claim 6, comprises identifying obstructive azoospermia by the presence of the haploid cells, diploid cells, and tetraploid cells in the testicular tissue.
9) The method as claimed in claim 6, comprising identifying meiotic arrest by the presence of the diploid cells and tetraploid cells and absence of the haploid cells in the testicular tissue.
10) The method as claimed in claim 6, comprises identifying premeiotic arrest by the presence of diploid cells and absence of the tetraploid cells and the haploid cells in the testicular tissue.
11) The method as claimed in claim 1, comprising determining a requirement for surgical sperm retrieval in the azoospermia patient.
12) The method as claimed in claim 1, comprising reproducibly determining the etiology of the azoospermia in the patient.
13) The method as claimed in claim 1 wherein the azoospermia includes both obstructive and non-obstructive azoospermia.
14) The method as claimed in claim 1, in the form of a diagnostic assembly or kit for rapid, accurate and economically viable screening of azoospermia in patients.
15) The method as claimed in claim 1, comprises a rapid, minimally invasive technique for assisting the clinicians in pre and post-surgical treatment of infertility.in the azoospermia patient, as a guiding technique for avoiding unnecessary surgeries.
16) A rapid, accurate, less-traumatic, minimally invasive DNA-flow cytometric method of analyzing azoospermia in patients afflicted with infertility due to physical, physiological, infective and medical conditions and azoospermia of unknown etiology for assisting the clinician in treating infertility conditions.
, Description:F O R M 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: METHODS AND DEVICES TO STUDY SPERMATOGENESIS IN HUMAN TESTICULAR TISSUES

2. APPLICANT (A) NAME: ANKUR ANDROLOGY & MENS HEALTH PRIVATE LIMITED

(B) ADSDRESS: B-701, RENAISAANCE TEMPLE BELLS, RENAISAANCE TEMPLE BELLS, BANGALORE, KARNATAKA, INDIA, 56002

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
[001] TECHNICAL FIELD OF THE INVENTION
[001] The present invention is in the technical field of flow Cytometry based identification of the haploid, diploid and tetraploid cell populations in a fine needle aspiration cytology (FNAC) sample. Based on the etiology and presence/absence of sperms, azoospermic patients can be diagnosed, for assisting the clinician in treating infertility conditions.
[002] BACKGROUND OF THE INVENTION
[003] Infertility is defined as the “inability of a sexually active, non-contraception couple to achieve pregnancy in one year” (WHO). There are an estimated 48.5 million couples with infertility worldwide, 30-50% of which are accounted by male factor infertility, a multifactorial disorder comprising a wide variety of clinical presentations. Female factor comprise approximately 40-45% of all infertility cases while idiopathic or unknown factors constitute about 5-8%.
[004] The clinical assessment of male infertility is primarily done through the analysis of semen (WHO, 2010). In addition to the semen parameters, the gonadotropin and androgen profile (free and total testosterone, estradiol, LH and FSH) are also typically used to assess the male reproductive function. The diagnosis of male infertility involves a detailed study of the following parameters: Complete medical history including duration of infertility:
[005] Scrotal ultrasound
[006] Hormone profiles
[007] Semen Analysis
[008] Microbiological evaluation of testes and the individual as a whole
[009] The WHO revised the parameters for semen analysis in 2010 establishing the reference values that were much lower than their previous ones, resulting in more men qualifying as normal (Cooper et at., 2010). The revised parameters are shown in the table below.
Semen Parameters Reference value (WHO, 2010) (Cooper et al., 2010)
Volume 1.5 ml
pH 7.0 – 8.0
Total sperm concentration 40 Million
Total sperm Motility 40%
Progressive motility 32%
Vitality 58%
Sperm Morphology 4%
Leukocyte count <1x106 / ml
[010] Based on the above mentioned parameters individual men can be grouped into different categories.
Type Semen Characteristics
Aspermia `No ejaculate
Azoospermia No sperm in the ejaculate
Oligozoospermia Sperm count less than 40 million
Asthenozoospermia Sperm motility less than 40%
Teratozoospermia Sperm morphology less than 4%
Combined Olig-Astheno-Teratozoospermia Sperm count <40, motility <40 and morphology <4%.
Normazoopermia Normal semen profile and infertility of unknown etiology

[011] Azoospermia is a type of male infertility associated with the absence of measurable spermatozoa in the semen, accounts for 20–30% of all male infertility cases affecting about 1% of the male population. Sperm retrieval from the testis, followed by Intracytoplasmic sperm injection (ICSI) is one of the procedures adopted for assisted reproduction. The common methods for sperm retrieval include testicular sperm aspiration (TESA), conventional testicular sperm extraction (TESE) and micro dissection TESE (m-TESE) and in several cases, the patients undergo multiple sperm retrievals.
[012] Sperm retrieval rates (SRR) vary depending upon the type of azoospermia with success rates of >90% in the case of obstructive azoospermia (OA) patients and it drops sharply to only about 30% in the case of non-obstructive azoospermia condition ( NOA). However, the success of sperm retrieval is dependent of the status of spermatogenesis in the testes of the individual and it is absolutely essential to investigate this status in the patients. Analysis of several pre-operative variables including follicle stimulating hormone (FSH) and lutenizing hormone (LH) levels, testicular size and volume have been employed to diagnose the type of azoospermia and predict the outcome of sperm retrieval. However, these factors have low sensitivity and specificity in predicting the success of sperm retrieval.
[013] A reliable post-intervention predictive method for successful sperm retrieval involves histological analysis of the testicular biopsies and this is considered one of the most preferred methods of diagnosis for establishing azoospermia condition in the patient with certainty. Histopathological analysis of the testicular biopsies requires proper fixation of the tissue to enable identification of all the cell types during spermatogenesis and an able pathologist who has a thorough understanding of the morphology of individual germ cell types can only recognize all cell types. The lack of a systemic approach, divergent reporting systems and the long time period required for the analysis can come in the way of diagnosis and clinical-care.
[014] Though several pre-clinical, non-invasive procedures have come to the fore in the recent years, it is still a long way to achieve high diagnostic accuracy and precision with such techniques. Of the two, NOA detection is more difficult and often challenging since there are no physical or physiological causating factors associated with it. Moreover, it turns out to be traumatic and burdensome for NOA patients when they have to undergo repeated surgical and invasive medical procedures with no positive outcome.
[015] The technology in the ART has various solutions for Oligo-Astheno-Terato, zoospermic conditions. However, in case of Azoospermia, the only solution is microsurgical way of finding sperms. Even after microsurgery, the clinician may or may not find the sperms based on the etiology of the patient. If the microsurgical evaluation may not yield any sperms resulting in waste of time, money and efforts with additonal emotional distress to the patient. To avoid this problem, in the present application we have designed and developed the flow cytometry based methodology to identify the presence of sperms in the azoospermic patient without the surgery.
[016] A single, rapid and reproducible diagnostic test to predict the type of azoospermia (especially in cases of NOA) is still not available.
[017] Thus, there is an urgent need to develop a rapid and simple method of investigating human spermatogenesis.
[018] In addition, there is also an urgent need to come out with an accurate test to know the stage at which the spermatogenesis is arrested.
[019] In the recent years some reports on DNA flow cytometric analyses of the germ cell populations for accurately and rapidly describing the status of spermatogenesis in the laboratory animal models including primates has been developed.
[020] Flow cytometry as a quantitative method for analyzing the testicular germ cells, flow cytometric evaluation of testicular germ cell transformations by specific hormonal deprivation or stimulation have been doing rounds in the research and academic circles.
[021] Patent number AU2018275005 disclosed a “Method of intracellular infectious agent detection in sperm cells”. Patent number WO/2017/088829 disclosed a “Methods for separating and culturing human testicular mesenchymal stem cells, and use of human testicular mesenchymal stem cells”. Patent 20140141420 disclosed “Method for successful retrieval of sperm”. In the paper titled “Seminal Haploid Cell Detection by Flow Cytometry in Non-Obstructive Azoospermia: A Good Predictive Parameter for Testicular Sperm Extraction “ Isabelle Koscinski et al Human Reproduction 20(7):1915-20 · August 2005, the authors’ noted that detection of seminal haploid cells by Flow-Cytometry Measurement appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients though it is less specific.
[022] However, none of the studies have given any conclusive evidence for employing such procedures in humans and especially on NOA patients.
[023] Hence, there is every need to come out with a reliable, accurate, rapid and non-invasive diagnostic method to analyze NOA in humans for obtaining conclusive results and at the same time mitigating the trauma and economic burden associated with the existing diagnostic procedures in the case of NOA.

[024] STATEMENT OF THE PROBLEM
[025] The present invention is taken up to come out with a less-traumatic and economical DNA-flow Cytometry based technique for the identification of spermatogenesis in azoospermia especially Non-ostructive azoospermia to accurately, rapidly and non-invasively predicting azoospermia in patients. The DNA-flow Cytometry based technique identifies the haploid, diploid and tetraploid cell populations in the FNAC sample obtained from the testis of azoospermic patients. In this method a single cell preparation of the germ cells from the FNAC tissue obtained is treated with propidium iodide, a nucleic acid binding dye. The amount of dye binding and its fluorescence intensity in flow Cytometry is directly dependent on the chromosome status i.e haploid, diploid or tetraploid. Based on the haploid, diploid and tetraploid populations present in the testis, the etiology and presence/absence of sperms in the testis of azoospermic patients can be understood.

[026] SUMMARY OF THE INVENTION
[027] According to embodiments of the present invention discloses a method of single, rapid and reproducible flow Cytometry based identification of the haploid, diploid and tetraploid cell populations in a fine needle aspiration cytology (FNAC) sample. Based on the etiology and presence/absence of sperms, azoospermic patients can be diagnosed. In addition, the method predict the type of azoospermia, outcome of sperm retrieval and assists clinician in treating infertility conditions.
[028] In one preferred embodiment, the present invention deals with a flow Cytometry based identification of the haploid, diploid and tetraploid cell populations in the FNAC sample obtained from the testis of azoospermic patients. In this method a single cell preparation of the germ cells from the FNAC tissue obtained is treated with propidium iodide, a nucleic acid binding dye. The amount of dye binding and its fluorescence intensity in flow Cytometry is directly dependent on the chromosome status i.e haploid, diploid or tetraploid. Based on the haploid, diploid and tetraploid populations present in the testis, the etiology and presence/absence of sperms in the testis of azoospermic patients can be understood (FIG 1). If the flow Cytometry analysis reveals the presence of the haploid, diploid and tetraploid cell populations, it can be concluded that spermatogenesis is complete in the patient’s testis and there is availability of sperms in the testis. If the flow Cytometry analysis reveals presence of the diploid and tetraploid cell populations, but not the haploid cells, it can be concluded that the spermatogenesis is arrested the at secondary spermatocyte stage (meiotic arrest). If the flow Cytometry analysis shows only the diploid cell population with no tetraploid and haploid cells. It indicates the arrest of the spermatogenesis before the primary spermatocyte formation (premeiotic arrest).
[029] In one more preferred embodiment of the invention, the technology contained in the invention identifies the presence of sperm in the testis on azoospermic patient with minimal invasive method (FNAC) and gives a clear indication for the clinician about the availability of the sperm. Based on the test results the clinician can decide further plan of action to go for surgery or not. Hence it avoids the unnecessary surgical procedure for sperm extraction (Micro-TESE) for the Non obstructive azoospermic patient. The financial burden and the actual pain suffered by the patient can be avoided to the maximum extent possible.
[030] In one embodiment flow cytometric test can be employed to rapidly establish the status of intra-testicular spermatogenesis in patients with azoospermia, as well as, to predict the exact reason for the arresting of spermatogenesis and also the stage at which the spermatogenesis is arrested with accuracy.
[031] In one more preferred embodiment the flow cytometric test performed with small amount of biopsy tissue is sufficient for diagnosis of spermatogenic disruption and the success of sperm retrieval from patients included in the study corroborated with their flow cytometric profile. The diagnostic technique prevented the trauma associated with multiple biopsies and atrophy due to multiple sampling as it happens with the existing methods like TESE.
[032] Most preferred embodiment of the invention discloses the method of analysis containing testicular tissue biopsy samples are collected from the azoospermic patients undergoing sperm retrieval prior to their surgical procedure. The tissue samples will be collected in Dulbecco’s Phosphate Buffered Saline (DPBS; Life Technologies Corporation, USA) with 0.5% glucose (Sigma Aldrich, USA) and transported on ice to the laboratory. To obtain single cell preparations of the testicular cells, the tissue samples will be suspended in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies Corporation, USA) and digested with 0.04% Collagenase (Type IV; Life Technologies Corporation, USA) and 15 µg/ml DNase-I (Sigma Aldrich, USA) at 32 °C in a shaking water-bath for 90 minutes. At the end of the incubation, the cells will be centrifuged at 100g for 10 min, washed in DPBS and a fraction of cell preparation will be fixed in 70% ethyl-alcohol (Merck Specialities Private Ltd. India) for flow cytometric analysis.
[033] In summary, the present invention discloses a single, rapid and reproducible diagnostic method to predict the type of azoospermia and outcome of sperm retrieval. The feasibility of employing DNA flow cytometry for rapid investigation of the status of spermatogenesis in the patients with azoospermia is disclosed in this invention. Testicular biopsies of 44 patients with azoospermia undergoing sperm-retrieval surgery and 4 controls were analyzed by flow cytometry to ascertain their testicular germ-cell patterns. The observed germ cell pattern was further confirmed by RT-PCR analysis of the cell-specific markers and histology for some patients. The patients with Obstructive Azoospermia (OA) exhibited normal spermatogenesis similar to the control fertile patients showing the presence of diploid, double-diploid and haploid cells. The non-obstructive azoospermia (NOA) patients exhibited disrupted spermatogenesis with arrest at the pre-meiotic (only diploid cells present) or meiotic (diploid and double-diploid cells present) stages. The germ-cell pattern, as ascertained by flow cytometry, provided a clear picture of the intra-testicular spermatogenesis and the presence of spermatozoa in the patients’ testes, which was prognostic of their sperm-retrieval. DNA flow cytometry test to ascertain the testicular germ-cell pattern is simple in execution, analysis and interpretation, requires small amount of tissue and provides quantitative data about the status of spermatogenesis in patients. This method would allow comparable analysis of the status of spermatogenesis in patients across clinics and may form the basis for deciding future treatment and intervention strategies.
[034] The present invention also relates to a method of detecting azoospermia using DNA-flow-cytometric technique in both obstructive and non-obstructive azoospermia conditions unknown etiology in a rapid, minimally invasive, accurate and economical way by investigating the spermatogenesis pattern of germ cells of the testes of humans and the various techniques employed to arrive at the azoospermia status and diagnostic kits associated with it and the various conditions under which the diagnostic methodology can be employed thereof.
[035] As will be appreciated by a person skilled in the art the present invention provides a variety of following embodiments. Listing of Claims,
[036] A method for analysing azoospermia, wherein the method comprises,
collecting a testicular tissue from a testis of a subject using a minimally invasive method;
performing a deoxyribonucleic acid (DNA) flow cytometric analysis on the collected testicular tissue;
identifying a chromosomal status of spermatogenesis in the testis based on the result of DNA flow cytometric analysis; and
determining etiology of the azoospermia in the patient based on the status of the spermatogenesis.
[037] The method as claimed in claim 1, wherein the minimally invasive method comprises obtaining the testicular tissue using a fine needle aspiration cytology (FNAC) method.
[038] The method as claimed in claim 1, comprising isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis.
[039] The method as claimed in claim 3, wherein performing the DNA flow cytometric analysis comprises isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis with a nucleic acid binding dye and analysing amount of dye binding and fluorescence intensity of the dye.
[040] The method as claimed in claim 4, wherein the nucleic acid binding dye comprises propidium iodide.
[041] The method as claimed in claim 1, wherein identifying the chromosomal status of spermatogenesis comprises identification of presence or populations of haploid cells, diploid cells, tetraploid cells, or combinations thereof.
[042] The method as claimed in claim 6, wherein determining etiology of the azoospermia in the patient comprises identifying obstructive azoospermia, meiotic arrest, or premeiotic arrest, based on the presence or populations of the haploid cells, diploid cells, tetraploid cells, or combinations thereof in the testicular tissue.
[043] The method as claimed in claim 6, comprises identifying obstructive azoospermia by the presence of the haploid cells, diploid cells, and tetraploid cells in the testicular tissue.
[044] The method as claimed in claim 6, comprising identifying meiotic arrest by the presence of the diploid cells and tetraploid cells and absence of the haploid cells in the testicular tissue.
[045] The method as claimed in claim 6, comprises identifying premeiotic arrest by the presence of diploid cells and absence of the tetraploid cells and the haploid cells in the testicular tissue.
[046] The method as claimed in claim 1, comprising determining a requirement for surgical sperm retrieval in the azoospermia patient.
[047] The method as claimed in claim 1, comprising reproducibly determining the etiology of the azoospermia in the patient.
[048] The method as claimed in claim 1 wherein the azoospermia includes both obstructive and non-obstructive azoospermia.
[049] The method as claimed in claim 1, in the form of a diagnostic assembly or kit for rapid, accurate and economically viable screening of azoospermia in patients.
[050] The method as claimed in claim 1, comprises a rapid, minimally invasive technique for assisting the clinicians in pre and post-surgical treatment of infertility.in the azoospermia patient, as a guiding technique for avoiding unnecessary surgeries.
[051] A rapid, accurate, less-traumatic, minimally invasive DNA-flow cytometric method of analyzing azoospermia in patients afflicted with infertility due to physical, physiological, infective and medical conditions and azoospermia of unknown etiology for assisting the clinician in treating infertility conditions.
[052] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
[053] BRIEF DESCRIPTION OF THE DRAWINGS
[054] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[055] FIG. 1 illustrates various phases of spermatogenesis, the organization of the germ cells with distinct cellular association into stages with chromosome numbers are shown.
[056] FIG. 2 illustrates the Germ cell patterns of the control and azoospermic patients. The representative flow cytometric profiles of the testicular biopsy samples as classified into different groups are shown. The testicular tissue samples from the control and azoospermic men were digested with collagenase and the single cell suspensions were fixed with 70% ethanol. The cells stained with PI were analyzed by flow cytometry. Panel A: control-C1-C4; Panel B: Group I – I a-d (obstructive azoospermia, exhibiting haploid, diploid and double-diploid populations); Panel C: Group II – II a-d (NOA with meiotic arrest, exhibiting diploid and double-diploid populations); Panel D: Group III– III a-d (NOA with pre-meiotic arrest, exhibiting diploid populations only according to embodiments of the invention.
[057] FIG. 3 illustrates expression of testicular cell specific marker genes. The expression status of the testicular cell specific markers for each representative patient samples as shown in the Fig. 2 is shown here. The total testicular RNA obtained from tissue samples was converted to cDNA by RT-PCR and the expression of the testicular cell specific markers was analyzed by semi-quantitative PCR using the specific primers. Each patient sample was analyzed individually by PCR for the expression of the testicular cell type specific markers as and when the sample was collected. Cropped images from individual gels are shown. C1–C4: control; I a-d: Group I (obstructive azoospermia, exhibiting haploid, diploid and double-diploid populations); II a-d: Group II - (NOA with meiotic arrest, exhibiting diploid and double-diploid populations); III a-d: Group III (NOA with premeiotic arrest, exhibiting diploid populations only) according to embodiments of the invention.
[058] FIG. 4 illustrates Histological Analysis of tissue samples representative for each group. 5 µm thick testicular tissue sections were stained with Haematoxylin and Eosin and observed under light microscope. The cell types are annotated as SC (the Sertoli cells), 1 (spermatogonia), 2 (spermatocytes) and 3 (round spermatids). Elongated spermatids are marked with black arrowheads. Panels A-B: I c, I d: Group I (obstructive azoospermia, exhibiting haploid, diploid and double-diploid populations); Panels C-F: II a-d: Group II - (NOA with meiotic arrest, exhibiting diploid and double-diploid populations); Panel G: III d: Group III (NOA with pre-meiotic arrest, exhibiting diploid populations only) (Scale bar = 20 µm, Magnification 40X) according to embodiments of the invention.
[059] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[060] . DETAILED DESCRIPTION OF THE INVENTION
[061] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology herein is for the purpose of description and should not be regarded as limiting.
[062] [002] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[063] The terms "subject", "patient" or "individual" generally refer to a human or mammals.
[064] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[065] IMPORTANT EMBODIMENTS OF THE SYSTEM:
[066] Spermatogenesis is a complex and coordinated process of formation of sperms from precursor spermatogonia. These unique processes of development can be divided in to three distinct phases.
[067] The proliferation of spermatogonia through mitosis
[068] Miosis or reduction division, which commences with transformation of the type B spermatogonia in to the primary spermatocytes and their subsequent entry in to the meiotic prophase 1st where upon they divide to form secondary spermatocytes, and then divide again to form haploid round spermatids.
[069] The successful differentiation of the round spermatids without further division to form the unique spermatozoa (spermatogenesis). The duration of the entire process of spermatogenesis varies from species to species ranging from 35 days in mice and Hamsters, 50 days in rats, 54 days in rabbits and 64-70 days in humans
[070] The various phases of spermatogenesis, the organization of the germ cells with distinct cellular association into stages with chromosome numbers are shown below in FIG 1.
[071] In the present application, inventors have developed a flow Cytometry based identification of the haploid, diploid and tetraploid cell populations in the FNAC sample obtained from the testis of azoospermic patients. In this method we obtain a single cell preparation of the germ cells from the FNAC tissue and treat it with propidium iodide which is a nucleic acid binding dye. The amount of dye binding and its fluorescence intensity in flow Cytometry is directly dependent on the chromosome status i.e haploid, diploid or tetraploid. Based on the haploid, diploid and tetraploid populations present in the testis, the etiology and presence/absence of sperms in the testis of azoospermic patients can be understood.
[072] 1] If the flow Cytometry analysis reveals presence of the haploid, diploid and tetraploid cell populations, it can be concluded that spermatogenesis is complete in the patient’s testis and there is availability of sperms in the testis.
[073] 2] If the flow Cytometry analysis reveals presence of the diploid and tetraploid cell populations, but not the haploid cells, it can be concluded that the spermatogenesis is arrested the at secondary spermatocyte stage (meiotic arrest).
[074] If the flow Cytometry analysis shows only the diploid cell population with no tetraploid and haploid cells. it indicates the arrest of the spermatogenesis before the primary spermatocyte formation (premeiotic arrest).
[075] Ethics Statement. The experiments described here were performed after obtaining appropriate clearances from the Institutional Review Board of the Manipal Ankur Fertility Clinic and the human ethics committee of the Indian Institute of Science (HIEC No: 10/10/2015). Informed consent was obtained from all subjects. All experiments were performed in accordance with the relevant guidelines and regulations with prior approval.
[076] Human patient samples. Testicular tissue biopsy samples were collected from the azoospermic patients undergoing sperm retrieval prior to their surgical procedure at the Manipal Ankur Fertility Clinic, Bangalore, India. The median age of patients was 32 years with the lower and upper hinges being 30 and 35 years respectively. The individual ages are provided in the Supplementary Table I. The characterization of all the human testicular tissue samples is summarized in the Table 1. Four tissue samples were also obtained from the fertile individuals undergoing testicular surgery for causes other than infertility (CA prostrate and orchidectomy for trauma). The tissue samples were collected in Dulbecco’s Phosphate Buffered Saline (DPBS; Life Technologies Corporation, USA) with 0.5% glucose (Sigma Aldrich, USA) and transported on ice to the laboratory. To obtain single cell preparations of the testicular cells, the tissue samples were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies Corporation, USA) and digested with 0.04% Collagenase (Type IV; Life Technologies Corporation, USA) and 15 µg/ml DNase-I (Sigma Aldrich, USA) at 32 °C in a shaking water-bath for 90 minutes. At the end of the incubation, the cells were centrifuged at 100 × g for 10 min, washed in DPBS and a fraction of cell preparation was fixed in 70% ethyl-alcohol (Merck Specialities Private Ltd. India) for flow cytometric analysis. The rest of the cells were resuspended in TRI Reagent (Sigma-Aldrich, USA) to extract RNA.
[077] Flow Cytometry Analysis. The ethanol fixed cells were washed twice with chilled DPBS, incubated with 20 µg/ml RNase-A (Sigma-Aldrich, USA) at 37 °C for 2 hours. The cells were stained with Propidium Iodide (PI;Sigma-Aldrich, USA) (100 µg/ml) for 30 minutes on ice and analyzed in BD FACSVerse (Becton Dickinson, USA)on the basis of their DNA content(1). The Peripheral Blood mononuclear cells (PBMC) isolated from the human blood were used as controls to fix the voltage for the diploid population and all samples were analyzed at this fixed voltage. The doublet population having high PI-area and PI-width was removed before data acquisition by gating on the PI width versus area dot-plot. The closely clustered populations were considered to be the haploid, diploid and double-diploid populations respectively based on the PBMC peak and are shown as interval gates on the histograms. The diploid cell population was fixed at 100 PI fluorescence intensity (peak-P1) and the haploid and double-diploid populations showed peaks at the half (50) (peak-P2) and double (200) (peak-P3) intensity respectively. Approximately 10,000 cells (excluding the dead cells and debris which were identified based on their forward and side scatters as well as low PI staining intensity) were analyzed for each patient sample. The azoospermic individuals exhibited an arrest of spermatogenesis or a mechanical blockage of the ductal systems leading to accumulation of spermatozoa in the epididymis. In many cases, the tubular architecture was compromised and increased cellular degeneration and death is expected. To reflect this observation, the final histograms shown include the dead cells and debris, though the dead cells were gated to allow the acquisition of 10,000 testicular cells.
[078] cDNA synthesis and semi quantitative PCR analysis. Total RNA was isolated from each patient’s sample with TRI reagent according to the manufacturer’s protocol. RNA (2 µg) was reverse transcribed into cDNA with random primers using the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. For the marker gene analysis, cDNA equivalent to 20 ng of RNA was amplified by PCR using the 2x PCR Master Mix (Thermo Fisher Scientific, USA). Various marker genes representative of different cell populations were analyzed using semi quantitative PCR (35 cycles, saturation analysis) for ascertaining the presence of a particular cell type in the testis. The KIT was chosen as the spermatogonial marker while CCNA, which controls G1-S transition was chosen as the double-diploid specific marker. In addition LDHC, the testis specific isoform, which is expressed initially in the leptotene-zygotene spermatocytes cells with maximum expression in the spermatids was also used as the double-diploid specific marker. PRM1, which is exclusively expressed in the post meiotic cells was chosen to confirm the presence of the haploid germ cells. FSHR was used to confirm the presence of the Sertoli cells while LHCGR and the enzymes involved in the testosterone biosynthesis pathway HSD3B2 and HSD17B3 were used to confirm the presence of the Leydig cells. The specific primers for each marker gene and amplicon size are listed in the Supplementary Table II. The housekeeping-gene RPL35 was used as the positive control.
[079] Histology. A portion of the tissue was fixed in Bouin’s Fixative and subsequently dehydrated, embedded in paraffin and sections of 5 µm thickness were prepared and mounted onto glass slides. For histology, the mounted sections were rehydrated by incubating in varying gradients of alcohol (100%, 80%, 70% and 50%) followed by water. The sections were stained with haematoxylin and Eosin according to Nalbandian et al.(2). The sections were again dehydrated, mounted in DPX and images were captured using a Zeiss microscope and processed by Zeiss Axiocam 4.3 software.
[080] Statistical Analysis. The data were collected independently for 2 variables – percentage of cells with varied DNA content (haploid, diploid and double-diploid) in the testes of infertile patients using flow cytometry and the status of the sperm retrieval from all patients after surgery. The percentage of each cell type in the flow cytometric test was correlated to the status of sperm retrieval by using both Pearson (RP) and Point Biserial (RPB) correlation coefficients and the difference in the means (mean percentage of cells when sperm retrieval was positive or negative) was ascertained using the Students t-test with Welch’s correction. Further, the flow cytometry test was analyzed for its sensitivity, specificity and predictive value using Fisher’s exact test and the binomial proportion and its confidence interval was also determined.
[081] Significant Results as part of the embodiments of the invention
[082] Profiling of testicular germ cell pattern of individual patients using Flow cytometry. The testicular tissues of the control and infertile patients were analyzed by flow cytometry and classified according to their cellular patterns (FIG 2). The germ cell pattern of the control group of patients with proven fertility exhibited three distinct peaks corresponding to the diploid, double-diploid and haploid cell populations. Fourteen patients(Group I), who suffered from OA, also showed three distinct germ cell peaks clearly indicating that spermatogenesis in these patients was complete and equivalent to the control group of patients. The 30 patients suffering from NOA were further divided into two groups based on their flow cytometric profile. The Group II comprising 24patients showed presence of the diploid and double-diploid cells, but there was complete absence of the haploid cells indicating meiotic arrest of spermatogenesis. The third group of patients (6 patients) showed presence of only the diploid cells with complete absence of the double-diploid and haploid cells indicating the pre-meiotic arrest of spermatogenesis. The characterization of all the human testicular tissue samples is summarized in the Table 1. The clinical diagnosis, the results of flow cytometric tests including percentage of each cell type and the outcome of sperm retrieval are provided in the Supplementary Table I. To confirm that the flow cytometric profile was a true representation of the testicular germ cell pattern, RT-PCR analysis for the cell population specific markers was performed for each patient.
[083] Group Type of Azoospermia
[084] Table 1. Characterization of testicular tissues of azoospermic patients. Note: All values are shown as mean ± SD. FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, mIU: milli-international units, CI: Confidence Intervals.
[085] [010] Analysis of various gene markers to confirm the germ cell patterns in the patients’ testes. Various marker genes representative of different cell populations (Sertoli cells, Leydig Cells, Spermatogonia, Spermatocytes/Double-diploid Cells and spermatids/Haploid cells) found in the human testes were analyzed using semi quantitative PCR. The cell type specific markers chosen are described in the Supplementary Table II.
[086] In the control and OA groups of patients, who showed presence of all three germ cell populations, the spermatogonial marker KIT, double-diploid specific markers CCNA and LDHC, and the haploid specific marker PRM1 were amplified confirming the presence of three populations of the germ cells. FSHR amplification confirmed the presence of the Sertoli cells while amplification of LHCGR and enzymes of the testosterone biosynthesis pathway HSD3B2 and HSD17B3 confirmed the presence of the Leydig cells. The Group II patients did not amplify PRM1, clearly indicating absence of the haploid cells while the Group III patients did not amplify CCNA1, LDHC and PRM1 confirming absence of the double-diploid and haploid cells. However, both Groups showed amplification of FSHR, LHCGR confirming presence of the Sertoli and Leydig cells in patients’ testes. Thus, a clear correlation was seen between the flow cytometric picture of testis and marker gene analysis (FIG 3).
[087] Table 2. Specificity and Sensitivity of the flow cytometric test. Note: TP: true positive, FN: false negative, FP:false positive: TN: true negative.
[088] Histological analysis of testicular tissues to confirm presence of different germ cells in patients’ testes. Testicular histology of some of the patients in each group was carried out and various cell types were identified as described by McLachlan et al. (FIG 4). The OA group of patients (Group I) exhibited a typical cellular picture comparable to the normal fertile individuals with presence of all three populations of the germ cells and the normal tubular structure, which is in agreement with the flow cytometric analysis of the testis. All cell types were present in the tubule including the Sertoli cells (SC), spermatogonia, spermatocytes, round spermatids as well as elongated spermatids (FIG 4A and B). The Group II patients showed complete absence of the haploid cells and the spermatozoa with accumulation of the double-diploid cells in the lumen of the tubules (FIG 4C–F). In the Group III tissues a clear lack of both haploid and double-diploid cells was observed, with only the Sertoli cells and a few germ cells being present inside the tubule (FIG 4G). All the different samples are histologically examined as per standard procedures.
[089] Statistical significance of the flow cytometry test. The percentage of each testicular cell type (haploid, diploid and double-diploid) in each testicular biopsy, as ascertained by flow cytometry, was correlated to the sperm retrieval from each individual patient. As the correlation was drawn between a continuous variable (percentage of cells) and a dichotomous variable (sperm retrieval), the Point Biserial correlation coefficient (RPB) was calculated and was observed to be the same as the Pearson correlation coefficient (RP). The percentage of the haploid cells showed a positive correlation (RPB: +0.89, RP: +0.8922, CI 95%: 0.8099 to 0.9401; p < 0.0001) to sperm retrieval, while the percentage of diploid cells showed a negative correlation (RPB: -0.78, RP: -0.7798, CI95%: -0.8743 to -0.6284; p < 0.0001). The double-diploid cells did not show any correlation to the status of sperm retrieval (RP B: +0.05, RP: +0.0513, CI 95%: -0.2494 to 0.3430; p = 0.743). The means of the percentage of the haploid and diploid cells from the patients with positive (MSR+) and negative (MSR-) sperm retrieval was significantly different. The MSR+ ± SEM was 40.86 ± 4.146 and MSR- ± SEM was 3.869 ± 0.4976 for the haploid cells with p value < 0.0001, while for the diploid cells the MSR+ ± SEM was 40.28 ± 4.299 and MSR- ± SEM was 78.54 ± 2.546 with p value < 0.0001. The specificity analysis for the flow cytometric test was carried out using the percentage of the haploid cells and the status of sperm retrieval. The specificity and sensitivity was calculated for a range of thresholds of percentage of the haploid cells ranging from 0–70%. The specificity and sensitivity were unchanged in the threshold-range of 13–22% of the haploid cells. Subsequently, the percentage of the haploid cells =20% was fixed as the threshold for positive sperm retrieval. In the flow cytometric test, a testicular biopsy sample from a patient diagnosed with OA showed a very low percentage of the haploid cells (10.78%; OA 10), but had successful sperm retrieval. This was annotated as the false negative. The detailed statistical analysis is shown in the Table 2. The significance of the percentage of haploid cells in predicting sperm retrieval was evaluated using Fisher’s exact test. The flow cytometric test showed high sensitivity (~92.86%) and specificity (~100%) (p value < 0.0001) in predicting the outcome of sperm retreival. The false positive rate (a = 1 - specificity) was 0% while the false negative rate (ß = 1 – sensitivity) was 7.14%. The binomial proportion for the same was 0.9773 and the CI95% was 0.8798–0.9944.
[090] Thus the flow cytometric test is highly specific and sensitive in predicting the outcome of sperm retrieval in azoospermic patients.
[091] The results described above clearly show that flow cytometric test can be employed to rapidly establish the status of intra-testicular spermatogenesis in patients with azoospermia, as well as, to predict the exact nature of the arrest in spermatogenesis. The test is highly sensitive and provides a quantitative picture of spermatogenesis using minimal amounts of tissue.
[092] The flow cytometric test performed with small amount of biopsy tissue was sufficient for diagnosis of spermatogenic disruption and the success of sperm retrieval from patients included in the study corroborated with their flow cytometric profile. All patients in the Group I had successful sperm retrieval while it was unsuccessful in the case of patients lacking the haploid cells indicated by their flow cytometric profile, which confirms the efficacy of this method.
[093] The present method provides a clear indication of the possibility of presence of the haploid cells and probable sperm retrieval in the first attempt, eliminating the need for multiple retrieval attempts for the patients with NOA. Further, the exact nature of disruption to the extent of early or late meiotic arrest can be ascertained by employing subsequent analysis of the cell type specific markers following flow cytometry. The transcript of LDHC (the testis specific isoform of lactate dehydrogenase) is expressed initially in the leptotene-zygotene spermatocytes cells with maximum expression in the spermatids. As seen in the study, LDHC amplified in 14 of the Group II patients but not in the other 10 patients. This is indicative of the nature of meiotic arrest (early or late) as the expression of LDHC varies in these patients depending on the stage at which the double-diploid cells are arrested. Also, as mentioned above, the nature of pre-meiotic arrest (spermatogonial arrest, SCO) can be further quantified using analysis of marker genes for the different diploid cell populations.
[094] In case of the patient T9 in this study, the double-diploid peak in the flow cytometry was very small that was clearly confirmed by the low number of spermatocytes seen in the histological section while in case of patients T4, T11 and T16, who exhibited broad double-diploid peaks, also showed presence of large number of the spermatocytes (FIG. 4).
[095] The flow cytometry test to predict the outcome of sperm retrieval has a positive predictive value of 100% and the negative predictive value ~96.77% that are highly significant (Table 2).
[096] The test is simple in execution, analysis and interpretation providing a rapid and reproducible method for quantifying spermatogenesis and can be performed across the clinics with comparable results. Further, the results of interpretation do not vary depending upon the experience of the analyst.
[097] The present treatment modality for the Non obstructive azoospermic patient is a surgical procedure for sperm extraction (Micro-TESE). The cost in terms of financial burden and the actual pain suffered by the patient is huge and even after this procedure, the clinician cannot guarantee the availability of sperms in the testicular tissue. Thus, it is a big financial and physical stress for the patient.
[098] Our novel technology identifies the presence of sperm in the testis on azoospermic patient with minimal invasive method (FNAC) and gives a clear indication for the clinician about the availability of the sperm. Based on our test results the clinician can decide further plan of action to go for surgery or not.
[099] This technique saves unnecessary surgery and financial distress to the patient. And gives clear idea to the clinician to decide about patient surgery.
[0100] According to embodiments of the present invention, invention relates to a method for analysing azoospermia, wherein the method comprises, collecting a testicular tissue from a testis of a human using a minimally invasive method; performing a deoxyribonucleic acid (DNA) flow cytometric analysis on the collected testicular tissue; identifying a chromosomal status of spermatogenesis in the testis based on the result of DNA flow cytometric analysis; and determining etiology of the azoospermia in the patient based on the status of the spermatogenesis.
[0101] The method is a minimally invasive method comprises obtaining the testicular tissue using a fine needle aspiration cytology (FNAC) method.
[0102] The said method is comprising isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis.
[0103] The said method comprises isolating single cell preparations of the germ cells from the testicular tissue for subjecting to DNA flow cytometric analysis with a nucleic acid binding dye and analysing amount of dye binding and fluorescence intensity of the dye.
[0104] The said method, wherein identifying the chromosomal status of spermatogenesis comprises identification of presence or populations of haploid cells, diploid cells, tetraploid cells, or combinations thereof, wherein determining etiology of the azoospermia in the patient comprises identifying obstructive azoospermia, meiotic arrest, or premeiotic arrest, based on the presence or populations of the haploid cells, diploid cells, tetraploid cells, or combinations thereof in the testicular tissue.
[0105] According to embodiments of the present invention, the method comprises identifying obstructive azoospermia by the presence of the haploid cells, diploid cells, and tetraploid cells in the testicular tissue, wherein the method comprises identifying meiotic arrest by the presence of the diploid cells and tetraploid cells and absence of the haploid cells in the testicular tissue, wherein it comprises identifying premeiotic arrest by the presence of diploid cells and absence of the tetraploid cells and the haploid cells in the testicular tissue.
[0106] According to embodiments of the present invention, the method comprises determining a requirement for surgical sperm retrieval in the azoospermia patient,
[0107] According to embodiments of the present invention, the method comprises reproducibly determining the etiology of the azoospermia in the patient.
[0108] According to embodiments of the present invention, the method comprises the azoospermia includes both obstructive and non-obstructive azoospermia.
[0109] According to embodiments of the present invention, the method is in the form of a diagnostic assembly or kit for rapid, accurate and economically viable screening of azoospermia in patients.
[0110] According to embodiments of the present invention, the method comprises a rapid, minimally invasive technique for assisting the clinicians in pre and post-surgical treatment of infertility.in the azoospermia patient, as a guiding technique for avoiding unnecessary surgeries.
[0111] A rapid, accurate, less-traumatic, minimally invasive DNA-flow cytometric method of analyzing azoospermia in patients afflicted with infertility due to physical, physiological, infective and medical conditions and azoospermia of unknown etiology for assisting the clinician in treating infertility conditions.
[0112] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0113] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0114] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[000] REFERENCE:
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2) Nalbandian, A., Dettin, L., Dym, M. & Ravindranath, N. Expression of vascular endothelial growth factor receptors during male germ cell differentiation in the mouse. Biology of Reproduction 69, 985–994 (2003).