Method for selecting an oocyte for implantation

Abstract

The present invention is directed to a method for determining suitability of an oocyte post intracytoplasmic sperm injection (ICSI) for embryo implantation including determining a ratio (q) between thickness of a zona pellucida (ZP) of the oocyte and an outer diameter of the oocyte, wherein a q value between a predetermined range is indicative of an embryo developed from the oocyte having increased implantation probability. The method further includes selecting the oocyte for implantation.

Description

METHOD FOR SELECTING AN OOCYTE FOR IMPLANTATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 652,151, titled "SYSTEMS, METHODS AND DEVICES FOR DETERMINING MECHANICAL RESPONSE OF THE HUMAN ZONA PELLUCIDA AND ITS INFLUENCE ON EMBRYO IMPLANTATION POTENTIAL DURING IN-VITRO FERTILIZATION TREATMENT", filed May 27, 2024, the contents of which are hereby incorporated by reference in their entirety.FIELD OF INVENTION

[0002] The present disclosure relates to intracytoplasmic sperm injection (ICSI) techniques, and more particularly to methods for assessing mechanical properties of the zona pellucida so as to predict embryo implantation potential.BACKGROUND

[0003] In vitro fertilization (IVF) has become an established method for assisting couples experiencing difficulties conceiving naturally. This assisted reproductive technology involves several stages, including ovarian stimulation, oocyte retrieval, fertilization, embryo culture, and embryo transfer. Despite advancements in IVF techniques, success rates remain relatively low, with pregnancy and live birth rates typically below 35% and 20% respectively per treatment cycle.

[0004] The human oocyte, or egg cell, consists of an inner cytoplasmic core containing cellular organelles and genetic material, surrounded by a thick elastic membrane known as the zona pellucida (ZP). ZP plays multiple roles in the fertilization process and early embryo development. It contains glycoproteins that act as receptors for sperm binding and facilitate sperm penetration. Additionally, the ZP helps prevent polyspermy (fertilization by multiple sperm cells) and protects the developing embryo until it is ready for implantation.

[0005] During IVF procedures, oocytes are typically retrieved from the ovaries and fertilized either through conventional insemination or intracytoplasmic sperm injection (ICSI).Following fertilization, the resulting embryos are cultured for several days before one or more are selected for transfer to the uterus. Throughout this process, embryologists assess various morphological and developmental parameters (morphokinetics) to help determine which embryos may have the highest potential for successful implantation and pregnancy.

[0006] One aspect of oocyte and embryo assessment that has received increasing attention is the mechanical properties of the zona pellucida. The ZP undergoes structural and biochemical changes during fertilization and early embryo development, a process sometimes referred to as "zona hardening." The ZP characteristics may influence the embryo's ability to hatch and implant in the uterine wall, which are necessary steps for establishing a pregnancy.

[0007] Current methods for evaluating embryo quality and selecting candidates for transfer rely primarily on morphological grading systems and time-lapse imaging of developmental milestones, which are known as morphokinetic parameters. While these approaches provide valuable information, they may not fully capture the complex factors that contribute to an embryo's implantation potential. There is ongoing interest in developing additional non- invasive assessment techniques that could complement existing selection criteria and potentially improve IVF outcomes.SUMMARY

[0008] According to the first aspect, there is provided a method for determining suitability of an oocyte post intracytoplasmic sperm injection (ICSI) for embryo transfer, the method comprising determining at least one geometrical parameter selected from the group consisting of: zona pellucida (ZP) thickness (t), ZP outer diameter (Dout), ZP inner diameter (Din), ZP average diameter (DaVerage (DOut+Din) / 2), suction length (L), any ratio thereof, and any combination thereof, wherein a value of the at least one geometrical parameter being between a predetermined range is indicative of an embryo developed from the oocyte having increased implantation probability compared to a control, thereby determining suitability of the oocyte post ICSI for embryo transfer.

[0009] According to another aspect, there is provided a method for selecting an oocyte post ICSI for embryo transfer, the embryo developing therefrom having increased probability of implantation compared to a control, the method comprising determining at least onegeometrical parameter selected from the group consisting of: zona pellucida (ZP) thickness (t), ZP outer diameter (Dout), ZP inner diameter (Din), ZP average diameter (Daverage (DOut+Din) / 2), suction length (L), any ratio thereof, and any combination thereof, wherein a value the at least one geometrical parameter, ratio thereof, or any combination thereof, between a predetermined range is indicative of an embryo developing from the oocyte having increased implantation probability compared to the control, and selecting the oocyte for embryo transfer.

[0010] In some embodiments, the method further comprises a step of selecting the oocyte for embryo transfer.

[0011] In some embodiments, the selecting comprises in vitro culturing the oocyte, cryopreserving the oocyte, transferring the embryo, or any combination thereof.

[0012] In some embodiments, the ratio is selected from the group consisting of: t / Dout, t / Din, t / Dout, t / Daverage, L / Din, L / Dout, L / Daverage, t / L, and any combination thereof.

[0013] In some embodiments, the at least one geometrical parameter, ratio thereof, or any combination thereof, is determined at a predefined pressure value.

[0014] In some embodiments, the at least one geometrical value being below or above the predetermined range, is indicative of an embryo developed from the oocyte having equal or reduced implantation probability compared to a control.

[0015] In some embodiments, the control comprises an oocyte the embryo developing therefrom failed to implant or did not properly implant.

[0016] In some embodiments, any one of: the transfer, and transferring is to a receptive female.

[0017] In some embodiments, the female is a human female subject.

[0018] In some embodiments, the method further comprises determining a shear modulus value (Cio) of the ZP of the oocyte based on the at least one geometrical parameter, ratio thereof, and any combination thereof.

[0019] In some embodiments, a Cio value ranging between 0.2-0.4 is indicative of the embryo developed from the oocyte having increased implantation probability compared to the control.

[0020] In some embodiments, the method is an in vitro or ex vivo.

[0021] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0022] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0023] Figs. 1A-1B include microscopic views of a human oocyte during an intracytoplasmic sperm injection (ICSI) procedure. Typical human oocyte (1A) and the two main stages of the ICSI procedure; Fixation and Injection (IB).

[0024] Figs. 2A-2F include microscopic view of sequential stages of embryo development during in-vitro fertilization. Embryo development stages: Fertilized oocyte (2A), Cleavage stage embryo (divisions into four distinct cells) (2B), Morula (2C), Expanded blastocyst with inner cell mass and trophectoderm (2D), Expanded blastocyst with ZP thinning (2E), and zona pellucida (ZP) rupture / hatching (2F).

[0025] Figs. 3A1-3D includes a flowchart of a methodology for analyzing oocyte images, including micrographs and graphs, according to aspects of the present disclosure. A flow chart of the methodology developed for study of ZP mechanical response: Images taken from the ICSI procedure (3A1-3A2), Image analysis to obtain oocyte specific geometry, aspiration length and fluid-air interface curvature in the holding pipette (3B1-3B2), Iterative computational finite element analysis of the fixation stage and computation of the pressure-aspiration length relationship (3C1-3C2), Comparison between observed and computed aspiration length to identify constitutive model parameters (3D).

[0026] Figs. 4A-4B include graphs showing oocyte measurements. Variation in outer diameter (4A) and ZP thickness (4B) for different oocytes from different patients.

[0027] Figs. 5A-5B include graphs showing oocyte measurements. Variation in outer diameter (5A) and ZP thickness (5B) for oocytes aspirated from the same patient. Circle mark the oocyte which was selected for embryo transfer without taking into account ZP mechanics (no pregnancy was obtained).

[0028] Fig. 6 includes illustrations of finite element models of ten different oocytes (2 oocytes from each patient).

[0029] Figs. 7A-7B include graphs and illustrations showing relationships between aspiration length (L, presented in pm) and material parameters for oocytes. Presented are computed L vs Cio curves for exemplary oocyte 3 of patient 1 (P1-O3; 7A) and P5-O5 (7B).

[0030] Fig. 8 includes a vertical bar graph showing the values of Cio for the ZP of the 10 different oocytes, before and after fertilization.

[0031] Fig. 9 includes a vertical bar graph showing the value of ZP hardening factor (ZPHF) for the different oocytes analyzed in the initial part of the study.

[0032] Fig. 10 includes graphs showing the relation between aspiration length L and Shear modulus for two different hypothetical strain energy-density functions (SEDFs).

[0033] Figs. 11A-11J include graphs comparing the computed L vs p curves obtained using the Neo-Hooken (NH), Mooney-Rivlin (MR) and Ogden model (OGDEN) SEDFs for aspiration length values observed in the clinical ICSI process (grey rectangle in each figure). The resulting range for each SEDF on the X axis (p [MPa]) is marked as follows: NH (1), MR (2), OGDEN (3).

[0034] Fig. 12 includes a surface plot showing oocyte mechanical properties. Manifold of Cio as a function of L and p obtained from computational mapping the parameter space of the oocyte fixation stage.

[0035] Fig. 13 includes a graph showing computed values of aspiration length L as a function of suction pressure for different values of Cio (different colored lines).

[0036] Figs. 14A-14B include vertical bar graphs showing a comparison between Cio values computed using oocyte specific modelling and values extracted from the manifold obtained by mapping the parameter space before fertilization (14A), and after fertilization (14B).

[0037] Fig. 15 includes a graph showing the value of Cio before fertilization for all embryos which were transferred into the patient. The black circles represent negative implantation while the empty circles represent positive implantation.

[0038] Fig. 16 includes a graph showing the value of Cio after fertilization for all embryos which were transferred into the patient. The black circles represent negative implantation while the empty circles represent positive implantation.

[0039] Fig. 17 includes a graph showing computed ZPHF for the different transferred embryos.DETAILED DESCRIPTIONMethods of determining and / or selecting

[0040] According to the first aspect, there is provided a method for determining suitability of an oocyte post intracytoplasmic sperm injection (ICSI) for embryo implantation.

[0041] According to another aspect, there is provided a method for selecting an oocyte post ICSI for embryo implantation, the embryo developing therefrom having increased probability of implantation compared to a control.

[0042] According to another aspect, there is provided a method for determining a shear modulus value of an oocyte during ICSI.

[0043] In some embodiments, the determining is of at least one geometrical parameter of the oocyte. In some embodiments, the at least on geometrical parameter is of the oocyte during ICSI. In some embodiments, the at least on geometrical parameter is determined for an oocyte during ICSI. In some embodiments, the at least on geometrical parameter is of the oocyte not more than 1 min, 10 min, 15 min, 30, or 60 min after ICSI, or any value and range therebetween. In some embodiments, the at least on geometrical parameter is determined for an oocyte not more than 1 min, 10 min, 15 min, 30, or 60 min after ICSI, or any value and range therebetween. In some embodiments, the method comprises determining the at least one geometrical parameter of the oocyte during or at the time of ICSI. In some embodiments, the methodcomprises determining the at least one geometrical parameter of the oocyte not more than 1 min, 10 min, 15 min, 30, or 60 min after ICSI, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0044] As disclosed herein, the invention, in some embodiments, provides accurate determination of suitability of an oocyte post intracytoplasmic sperm injection (ICSI) for embryo transfer, during ICSI. The method of the invention is advantageous and superior to current practice which is restricted to real-time live observation of embryo development, i.e., required culturing the oocyte post ICSI till embryo stage, hatching, or both. The common practice is clearly prolonged, laborious, and tedious. In sharp contrast, the method of the invention provides a fast ‘on the spot’ prediction with high specificity and accuracy.

[0045] Further, the method of the invention, in some embodiments, reduces the destruction of embryos. Specifically, current practice selects embryo for transfer based developmental parameters. In sharp contrast, the method according to some embodiments, provides early identification of oocytes post ICSI which are suitable for transfer (to a receptive female), but before an embryo is conceived. Thus, the method, in some embodiments, reduces dramatically or may even eliminate the destruction of embryos.

[0046] As used herein, the term "embryo" may refer to the early stages of development of a multicellular organism. In the context of assisted reproductive technologies, as described herein, an embryo is a multicellular organism that develops from the zygote through cell division and differentiation. To this, a zygote, i.e., a single-celled fertilized egg, is not an embryo. Further, an embryo may specifically refer to the developing multicellular organism, i.e., including 2 cells or more, after the first time the zygote began to divide and until the blastocyst stage, which occurs around day 5-6 post-fertilization. The term may encompass various stages of early development, including the cleavage stage, morula, and blastocyst. As would be apparent to a person of skill in the art, an oocyte during or immediately post ICSI is single-celled fertilized egg, i.e., zygote, which is not to be mistaken with an embryo, i.e., a multicellular organism including at least 2 cells.

[0047] In some embodiments, an oocyte during or post ICSI as disclosed herein, is a zygote as long as it has not undergone at least one complete cell division. In some embodiments, an oocyte during or post ICSI as disclosed herein, is not an embryo. In some embodiments, anoocyte during or post ICSI as disclosed herein, comprise a single cell. In some embodiments, an oocyte during or post ICSI as disclosed herein, does not comprise more than one cell.

[0048] In some embodiments, the at least one geometric parameter is selected from: zona pellucida (ZP) thickness (t), ZP outer diameter (Dout), ZP inner diameter (Din), ZP average diameter (DaVerage (DOut+Din) / 2), suction pressure (P), suction length (L), a ratio thereof, or any combination thereof.

[0049] In some embodiments, the ratio is selected from: t / Dout, t / Din, t / Dout, t / D average, L / Din, L / Dout, L / Daverage, t / L, or any combination thereof.

[0050] In some embodiments, the ratio is determined at a predefined pressure value.

[0051] In some embodiments, the predefined or predetermined (used herein interchangeably) pressure value is at least 0.001 mPa, at least 0.002 mPa, at least 0.003 mPa, at least 0.004 mPa, at least 0.005 mPa, or any value and range therebetween. In some embodiments, the predefined or predetermined (used herein interchangeably) pressure value ranges between 0.001 and 0.002 mPa, 0.001 and 0.003 mPa, 0.001 and 0.004 mPa, 0.001 and 0.005 mPa, 0.002 and 0.003 mPa, 0.002 and 0.004 mPa, 0.002 and 0.005 mPa, 0.003 and 0.004 mPaO.OOl, 0.003 and 0.005 mPa, or 0.004 and 0.005 mPa. Each possibility represents a separate embodiment of the invention.

[0052] In some embodiments, the predetermined range of t ranges between 5 and 30 pm, 6 and 28 pm, 9 and 26 pm, 5 and 27 pm. Each possibility represents a separate embodiment of the invention.

[0053] In some embodiments, the predetermined range of Din ranges between 70 and 200 pm, 80 and 180 pm, 85 and 175 pm, 75 and 170 pm. Each possibility represents a separate embodiment of the invention.

[0054] In some embodiments, the predetermined range of Dout ranges between 100 and 200 pm, 110 and 190 pm, 120 and 190 pm, 130 and 190 pm, or 140 and 180 pm. Each possibility represents a separate embodiment of the invention.

[0055] In some embodiments, the predetermined range of t / Dout ranges between 0.05 and 0.5, 0.05 and 0.15, 0.06 and 0.2, or 0.05 and 0.3. Each possibility represents a separate embodiment of the invention.

[0056] In some embodiments, the predetermined range of t / Dint ranges between 0.05 and 0.5, 0.05 and 0.18, 0.07 and 0.2, or 0.08 and 0.17. Each possibility represents a separate embodiment of the invention.

[0057] In some embodiments, the predetermined range of t / Daverage ranges between 0.05 and 0.5, 0.05 and 0.18, 0.06 and 0.2, or 0.06 and 0.17. Each possibility represents a separate embodiment of the invention.

[0058] In some embodiments, the predetermined range of L ranges between 1 and 50 pm, 1 and 50 pm, 1 and 40 pm, 1 and 35 pm, 5 and 50 pm, 5 and 40 pm, or 5 and 35 pm. Each possibility represents a separate embodiment of the invention.

[0059] In some embodiments, the predetermined range of L / Din ranges between 0.01 and 0.3, 0.01 and 0.2, 0.02 and 0.3, 0.03 and 0.3, 0.04 and 0.3, 0.04 and 0.3, 0.02 and 0.25, or 0.01 and 0.25. Each possibility represents a separate embodiment of the invention.

[0060] In some embodiments, the predetermined range of L / Dout ranges between 0.02 and 0.3, 0.02 and 0.25, 0.03 and 0.3, or 0.03 and 0.25. Each possibility represents a separate embodiment of the invention.

[0061] In some embodiments, the predetermined range of L / DaVerage ranges between 0.03 and 0.3, 0.03 and 0.25, 0.035 and 0.25, 0.04 and 0.25, or 0.04 and 0.3. Each possibility represents a separate embodiment of the invention.

[0062] In some embodiments, the predetermined range of t / L ranges between 0.1 and 10, 0.15 and 8, 0.2 and 10, 0.2 and 7, or 0.2 and 6. Each possibility represents a separate embodiment of the invention.

[0063] Means and methods for determining thickness of a zona pellucida (ZP), an outer diameter, inner diameter, average diameter, or any combination thereof, of an oocyte, are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for such methods of determination include, but are not limited to, Light microscopy imaging and measurement, Electron microscopy imaging and measurement, Confocal microscopy imaging and analysis, Optical coherence tomography (OCT) imaging, Ultrasound imaging and measurement, Micromanipulation techniques with calibrated micropipettes, Atomic force microscopy (AFM) measurements, Digital image analysis software applied to microscopyimages, Fluorescence microscopy with labeled zona pellucida proteins, Laser scanning confocal microscopy with 3D reconstruction, Phase contrast microscopy imaging and measurement, Differential interference contrast (DIC) microscopy, Polarized light microscopy, Raman spectroscopy imaging, Quantitative phase imaging techniques, Machine learning algorithms applied to oocyte images for automated measurements, Holographic imaging and analysis, Microfluidic devices with integrated optical measurement systems, Interferometric techniques for high-precision thickness measurements, Time-lapse imaging systems with automated measurement capabilities, to name a few.

[0064] In some embodiments, the determining is in real time. In some embodiments, the determining is during ICSI. In some embodiments, the method comprises determining the at least one geometric parameter, ratio thereof, or any combination thereof, during or simultaneously with the ICSI (performance). In some embodiments, real time refers to ICSI performance.

[0065] In some embodiments, the method comprises selecting the oocyte for embryo transfer. In some embodiments, the method further comprises a step of selecting the oocyte for embryo transfer.

[0066] In some embodiments, selecting comprises in vitro culturing, cryopreserving, transferring, or any combination thereof, the oocyte or the embryo developing therefrom.

[0067] Methods for in vitro culturing an oocyte or an embryo are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for methods of in vitro culturing include, but are not limited to, Static culture in microdroplets under mineral oil, Group culture in microwell dishes, Sequential culture media systems, Single-step culture media systems, Co-culture with somatic cells (e.g. cumulus cells, granulosa cells), Three-dimensional culture systems using hydrogels or scaffolds, Microfluidic culture devices with controlled media flow, Time-lapse imaging incubators for continuous monitoring, Low-oxygen tension culture conditions, Supplementation with growth factors or hormones, Addition of antioxidants to culture media, Use of embryo-conditioned media, Metabolomic profiling -guided media formulations, Temperature-controlled culture platforms, pH-stabilized culture systems, Mechanical stimulation during culture (e.g., tilting platforms), Hypoxia-inducible factor (HIF) stabilization culture conditions, Mitochondrial transfer or supplementation techniques, RNAinterference-mediated gene modulation during culture, Epigenetic modifiers added to culture media, to name a few.

[0068] Methods for cell cryopreservation are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for cell cryopreservation methods include, include but are not limited to, Slow freezing with controlled-rate freezers, Vitrification using high concentrations of cryoprotectants, Programmable freezing with stepwise cooling protocols, Directional freezing techniques, Encapsulation in alginate or other hydrogels before freezing, Use of intracellular and extracellular cryoprotective agents, Equilibrium freezing methods, Non-equilibrium freezing methods, Droplet vitrification, Cryoloop vitrification, Solid surface vitrification, Minimum volume vitrification, Closed system vitrification, Open system vitrification, Cryopreservation using magnetic freezing, Freeze-drying (lyophilization) for long-term storage, Isochoric freezing, Oscillating temperature freezing, Cryopreservation using ice-nucleating agents, Cryopreservation with antifreeze proteins or synthetic ice blockers, to name a few.

[0069] Methods for embryo transfer or implantation are common and would be apparent to one of ordinary skill in the art. Non-limiting examples for methods for embryo transfer or implantation, include but are not limited to, Transcervical embryo transfer using a soft catheter, Ultrasound-guided embryo transfer, Hysteroscopic embryo transfer, Transmyometrial embryo transfer, Tubal embryo transfer, Zygote intrafallopian transfer (ZIFT), Assisted hatching prior to transfer, Blastocyst culture and transfer, Time-lapse imaging for embryo selection before transfer, Preimplantation genetic testing for aneuploidy (PGT-A) before transfer, Endometrial receptivity analysis (ERA) for timing transfer, Use of embryo glue or adherence compounds during transfer, Sequential or double embryo transfer, Natural cycle embryo transfer, Hormone-replaced frozen embryo transfer, Intrauterine platelet-rich plasma (PRP) treatment before transfer, Endometrial scratching prior to transfer, Laser-assisted embryo transfer, Robotic-assisted embryo transfer, Magnetic-activated embryo transfer, to name a few.

[0070] In some embodiments, the at least one geometrical value, ratio thereof, or any combination thereof, being below or above the predetermined range, is indicative of an embryo developed from the oocyte having equal or reduced implantation probability compared to a control.

[0071] In some embodiments, a control comprises a control oocyte and / or an embryo developing therefrom. In some embodiments, a control comprises an oocyte, the embryo developing therefrom failed to implant or did not properly implant.

[0072] In some embodiments, implant, implantation, or implanting is in a female uterus. In some embodiments, a female is a human female subject, e.g., a woman. In some embodiments, the female is undergoing a fertility treatment or a plurality thereof. In some embodiments, transfer or transferring is to a female uterus. In some embodiments, a female is a human female subject, e.g., a woman. In some embodiments, the female is undergoing a fertility treatment or a plurality thereof. The terms “implant” or “implanting” and “transfer” or “transferring” are used herein interchangeably.

[0073] As used herein, the term "plurality" refers to any integer being equal to or greater than 2.

[0074] In some embodiments, the method further comprises calculating or determining an approximate aspiration length (L) of the oocyte based on the ratio between the oocyte thickness and diameter. In some embodiments, an approximate aspiration length (L) is suitable for determining a shear modulus value (CIO) of the ZP of the oocyte, as exemplified herein below.

[0075] As used herein, the term "aspiration length (L)" refers to the distance that the zona pellucida and / or oocyte membrane is drawn into a holding pipette during the fixation stage of an intracytoplasmic sperm injection (ICSI) procedure. This length may be measured from the tip of the holding pipette to the furthest point of deformation of the oocyte membrane within the pipette. The aspiration length may be influenced by factors such as the applied suction pressure, the mechanical properties of the zona pellucida, and the overall geometry of the oocyte.

[0076] As used herein, the term "approximate" refers to a value that is close to or near an exact value, but not necessarily identical. In the context of measurements or calculations, an approximate value may be within a certain percentage or range of the exact value, typically allowing for minor variations, experimental error, or computational limitations. The degree of approximation may depend on the specific context and requirements of the application or analysis being performed.

[0077] In some embodiments, the approximate length (L) is determined by calculation. In some embodiments, the approximate length (L) is not a measured length (L). In this regard, the construction of the L vs Cio curve for one oocyte which is necessary for determining the ZP shear modulus can take many hours (18-24 hours using parallel computing on 8 cores on a typical workstation). In sharp contrasty, the claimed invention provides the means to expedite the process of determining the ZP shear modulus, as it relies on rapid and accurate approximation of the length (L), which is determined during the ICSI or adjacent thereto, i.e., at the oocyte / zygote stage, and not once an embryo is obtained. Therefore, unlike the common practice known to a person of skill in the art which deals with embryo selection, the method of the invention, in some embodiments, allows rapid and accurate determination of the suitability of an oocyte undergoing ICSI for subsequent transfer, at the point of care or adjacent thereto, i.e., at the oocyte / egg / zygote stage.

[0078] In some embodiments, the method further comprises determining the shear modulus value (Cio) of the ZP of the oocyte based on the at least one geometric parameter, a ratio thereof, or any combination thereof.

[0079] In some embodiments, Cio ranging between 0.2-0.4 is indicative of an embryo to be developed from the oocyte has increased implantation probability compared to a control.

[0080] As used herein, the term "shear modulus" refers to a measure of a material's resistance to shear deformation. It represents the ratio of shear stress to shear strain in a material. In the context of zona pellucida mechanics, the shear modulus may characterize the elastic response of the zona pellucida to applied forces during procedures such as intracytoplasmic sperm injection or embryo development. The shear modulus may be denoted by symbols such as G or p and is typically expressed in units of pressure, such as pascals (Pa) or megapascals (MPa).

[0081] As used herein, the terms "zona pellucida" or "ZP" refer to a glycoprotein layer surrounding mammalian oocytes and early embryos. This extracellular matrix forms a protective envelope around the oocyte and plays crucial roles in fertilization and early embryonic development. The zona pellucida may be composed of several glycoproteins that contribute to its structural and functional properties. In human oocytes, the zona pellucida may typically range from 10 to 30 micrometers in thickness and may undergo biochemical and mechanical changes during oocyte maturation, fertilization, and embryo development. Thezona pellucida may be involved in processes such as sperm binding, prevention of polyspermy, protection of the early embryo, and regulation of embryo hatching prior to implantation.

[0082] As used herein, the terms "intracytoplasmic sperm injection" or "ICSI" refer to a specialized in vitro fertilization technique where a single sperm is directly injected into the cytoplasm of a mature oocyte. This procedure may be performed under microscopic visualization using micromanipulation equipment. ICSI may be used to overcome various forms of male infertility or in cases where conventional in vitro fertilization (IVF) techniques have been unsuccessful. The process may involve immobilizing the oocyte with a holding pipette, selecting a single sperm, immobilizing it, and then injecting it through the zona pellucida and oolemma into the oocyte's cytoplasm using a fine glass needle. Following injection, the oocyte may be monitored for signs of fertilization and subsequent embryonic development.General

[0083] 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, and are 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.

[0084] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0085] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0086] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0087] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0088] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0089] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0090] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Background

[0091] In Vitro Fertilization (IVF) is an accepted method for aiding couples who are unable to naturally conceive. There are many different reasons that can lead to infertility. These can be related to either male infertility, female infertility, a combined infertility or unexplained infertility. Part of the fertility issues are based on the quality of the sperm or the oocyte (human "egg"). Even though IVF treatments are well established, the current success rate for pregnancy and live birth rates are still low (35% and 20% respectively). The oocyte is composed of an inner core, known as the cytoplasm, containing the cell organelles and genetic material, and it is surrounded by a thick elastic membrane called the Zona Pellucida (ZP). The ZP contains glycoproteins that serve as receptors for sperm binding and participate in sperm penetration. ZP glycoproteins also have a role in preventing polyspermy (penetration of more than one sperm) and protecting the growing embryo till hatching and implantation. Based on the crucialrole of ZP, morphological and mechanical assessments of ZP are important for the determination of oocyte’s quality and embryo implantation potential.

[0092] On average, around eight oocytes are aspirated in each IVF cycle, depending on the woman's age and ovarian reserve. In cases of male infertility (poor sperm parameters), a procedure called Intra Cytoplasmic Sperm Injection (ICSI) is conducted. In this procedure a sperm cell is injected into each mature oocyte under a microscope.

[0093] The sperm injection during the ICSI process can be divided into two main stages: (1) Fixation of the oocyte using a pipette to apply suction which holds the oocyte in a proper position. (2) Injection of the sperm into the oocyte using a fine needle.

[0094] A typical oocyte with relevant dimensions and a basic outline of the ICSI procedure is shown in Fig. 1. It is important to note that during the fixation stage (center image in Fig. 1) of the procedure, the ZP is deformed into the holding pipette. The amount of deformation is termed the aspiration length.

[0095] Following ICSI, the oocytes are monitored for fertilization and allowed to develop into preimplantation embryos. During development, the embryos are monitored using an incubator fitted with a camera and external viewer (time lapse monitoring system -Embryoschope). Embryos are monitored using morphokinetic parameters that combine the morphological (shape) appearance and the kinetics of the cell’s divisions. Throughout the first three days the inner mass (cytoplasm), undergoes several divisions (cleavage stage) with no increase in the cell's volume. On the fourth day the cells undergo compaction and a morula is formed. On the fifth day the morula is transformed into a blastocyst in which the cells are differentiated into two types; 1) the inner cell mass which will form the embryo and 2) the trophectoderm which will form the placenta. Morphological changes in the trophectoderm result in expansion of the blastocyst. Finally, usually one or two embryos are selected by the embryologist to be transferred to the patient's uterus; this stage of embryo selection is termed the selection stage. At the blastocyst stage the embryo is subjected to a phenomenon called ‘hatching’ which includes its expansion with stretching, thinning and focal rupture of the ZP and at the end of the process, extrusion of the embryo through the fractured zona (that usually occurs inside the uterus). The hatching of the blastocyst is in fact a fundamental step for the establishment of pregnancy as it allows the blastocyst to adhere to the endometrium (uterine wall) and invadeit. If hatching does not occur the blastocyst cannot implant, and pregnancy does not take place. The different stages of embryo development from the fertilized oocyte to the hatching of the blastocyst are shown in Fig. 2.

[0096] It should be noted that the blastocyst expansion resulting in ZP thinning (Figs. 2D-2F) takes several hours. Surprisingly, even though the hatching process is at least partly a mechanically driven process, ZP mechanics currently does not play a role in embryo selection. The overall goal of the proposed research is to determine what specific values of human oocyte ZP mechanical parameters are required to attain a successful hatching following an ICSI procedure.

[0097] The clinical ICSI procedure conducted on the human oocytes, which were analyzed in this study, cannot be altered in any way due to ethical considerations. Therefore, the process of determining the ZP material parameters from the ICSI clinical data is based on analysis of images taken from the microscope and subsequent finite element analysis in conjunction with a minimization process of a target function. The computational process for determination of the ZP model parameters is conducted in several steps: (1) Images of each oocyte geometry undergoing the ICSI process are taken at various stages of the process. This includes the undeformed shape of the ZP before oocyte's holding, deformed shape of the ZP during oocyte holding by peptide suction with specific emphasis on the aspiration length and on the liquid air interface curvature in the peptide during the holding process. (2) For each oocyte geometry, a specific finite element (FE) model is constructed, and pressure boundary conditions are applied following analytical estimation of the pressure gradient across the liquid air interface. (3) An iterative process of minimizing a target function for the error in computed aspiration length L is conducted. (4) Once the iterative process is terminated, the ZP material model parameters that resulted in the measured aspiration length are identified. The methodology is outlined in Fig. 3.

[0098] Current data from ICSI procedures on 29 patients and 290 oocytes was collected. Initially a subset of 10 oocytes from 5 different patients (2 from each patient) were selected for analysis. This subset of oocytes was used to construct oocyte specific finite element models to study the different aspects of the ZP mechanics. These include the influence of oocyte geometry, changes in ZP shear modulus before and after fertilization and the sensitivity of theconstitutive model used to model the ZP mechanical response. Next, the insights gained from the first part of the study, on the subset of oocytes, were used to computationally map the parameter space of the oocyte fixation stage. This mapping process greatly reduces the time required to identify the ZP shear modulus both before and after fertilization. Finally, the ability of the ZP shear modules to predict implantation into the uterus (meaning that a successful hatching occurred) was examined. The following examples describe the results for all the above-mentioned research stages.EXAMPLE 1Collection of oocyte data from clinical ICSI procedures

[0099] Follicles were aspirated and the cumulus-oocyte complexes were delivered to the laboratory. After washing in Global Total with HEPES medium oocytes were cultured in fertilization medium covered with mineral oil for 3 h at 37 °C, 5.7% CO2, and 5% O2. Oocyte denudation was initiated by a 30-sec incubation in 80 lU / mL of hyaluronidase, followed by three washings in HEPES medium to remove residuals of the enzyme. The oocytes were then incubated in fertilization medium for an additional hour before denudation. Removal of cumulus cells from the oocyte was carried out by mechanical pipetting in Global Total medium containing HEPES. ICSI procedures were performed in the same medium at 400x magnification using a Nikon Eclipse Ti microscope.

[0100] Oocyte geometrical parameters ZP thickness and oocyte diameter were measured from digital images taken at three different orientations. These oocyte images, taken before the ICSI procedure and after fertilization, were analyzed using an in-house MATLAB code, which identified the boundary of the cytoplasmic core and the ZP. The boundary data were imported into a computer-aided design (CAD) program as a point cloud, and specific 2D and 3D models of the oocyte were generated.EXAMPLE 2Geometric variability of oocytes outer diameter and ZP thickness

[0101] Currently, a total of 63 (out of 290) oocytes from 27 (out of 29) patients which participated in the study were analyzed. Initial analysis consisted of using an In-house MATLAB code and image analysis tools to measure the ZP thickness and oocyte diameter. InFig. 4 the variations in oocyte diameter and ZP thickness are shown. The average oocyte outer diameter D was 162 ± l l[pm] and the average ZP thickness was 19 ± 3.35 [gm]. Estimated error in measurement of diameter and thickness using the Image processing tools was ±1 [pm]. As can be seen in Fig. 4, a great variance exists in both diameter and thickness with D ranging from 135 to 195 [pm] and t ranging from 11 to 26 [pm]. The variance in geometric parameters of the oocytes is large even when considering oocytes aspirated from the same patient as can be seen in Fig. 5.EXAMPLE 3Oocyte specific finite element modelling for determination of the ZP mechanical parameters before and after fertilization

[0102] In the initial part of the study, the geometric parameters of 10 oocytes taken from 5 different patients were utilized to construct oocyte specific finite element models for simulation of the fixation stage of the ICS I process. Following the methodology described by the authors in [2], the aspiration length L (Fig. IB) of the ZP into the pipette was computed. Because the ZP undergoes large deformations during the "hatching" process and also during the fixation stage of the ICSI procedure (in the suction region) a hyper-elastic constitutive model is necessary for modeling the ZP mechanical response. As in a previous study, a compressible Neo-Hookean strain energy density function was used:(1) .v / / (7c, / / / C)=CIO( / C1 / 3-3)+1 / I( / / / C1 / 2-1)2

[0103] With Ic and IIIc being the first and third invariants of the Right Cauchy deformation tensor and Cio=p / 2 and DI=2 / K representing the shear modulus p and bulk modulus K respectively. It was previously demonstrated that the aspiration length L is sensitive to the value of CIO but not sensitive to the value of DI. Therefore, a comparison between computed and observed aspiration length L can be used to identify the value of CIO, but not the value of DI. The different oocyte geometries for the first set of analysis can be seen in Fig. 6. The computation was repeated using different values of the material model parameter CIO which represents the ZP shear modulus. This enabled the generation of L vs CIO curves for each oocyte geometry as seen in Fig.7. Using the computed L vs CIO curves and the measured aspiration length LICSI obtained from the clinical data of the ICSI procedure, it is possible to determine the CIO value for the ZP of the specific oocyte both prior to fertilization andfollowing fertilization. In Fig. 8 the value of CIO before and after fertilization for the 10 oocytes analyzed in the initial part of the study are shown. It can clearly be seen that the value of CIO increases following the oocyte fertilization. This finding is consistent with a biological phenomenon termed Zona-Hardening. Here, the inventors define the ratio between CIO after fertilization to CIO before fertilization as the Zona Pellucida Hardening Factor (ZPHF). The value of the ZPHF for the different oocytes is given in Fig. 9. As can be seen in Fig. 9, the value of ZPHF from the different oocytes ranges from 1.1 to 1.77 with an average value of ZPHF of 1.535.EXAMPLE 4Investigating the influence of ZP constitutive model on shear modulus aspiration length relationship

[0104] In the study, the human oocyte ZP mechanical response is modeled using a compressible Neo-Hookean SEDF. The computational methodology for determining the ZP shear modulus (CIO value) is based on comparing the measured aspiration length L, to the computed L vs CIO curves shown previously (Fig. 7). For the Neo-Hookean model, the value of CIO represents the ZP shear modulus (see Eq. 1). Other forms of SEDFs can result in a different functional relationship between the aspiration length L and some material model parameter which represents the shear modulus. For example, in Fig. 10, the computed aspiration length L vs shear modulus for two hypothetical SEDFs are shown.

[0105] During the ICSI procedure the suction pressure is not monitored but a typical range exists for the observed aspiration length. As demonstrated in Fig. 10, for constitutive model B measurements of the aspiration length L are all related to a narrow range of shear modulus values. On the other hand, for constitutive model A, measurements of the aspiration length L are related to a greater range of shear modulus values. This makes constitutive model B match more sensitive to errors in measurements of the aspiration length compared to model A. In order to examine the sensitivity of the constitutive model for the purpose of identifying the ZP shear modulus, two additional SEDF's were considered.

[0106] The Mooney-Rivlin SEDF:With Ic, lie and IIIc being the invariants of the Right Cauchy deformation tensor and p=Cio+Coi, DI=2 / K representing the shear modulus p and bulk modulus K respectively.The Ogden SEDF:

[0107] With Ai being the deviatoric principal stretch ratios, where a, p (shear modulus) and DI=2 / K are material model parameters.

[0108] The computational analysis for the 10 oocytes shown in Fig. 6, was performed using the Neo-Hooken model (NH), the Mooney-Rivlin model (MR) and the Ogden model (OGDEN). Fig. 11 compares the sensitivity of all three constitutive models in the range of the aspiration length L observed in the clinical ICSI process for these 10 oocytes (L=15-25 [pm]).

[0109] The comparison revels that in all cases the MR model (Green rectangles in Figure 11) displays the narrowest range of shear modulus value. This makes the MR more sensitive to any errors in aspiration length measurements. Therefore, the MR model is less suited for modeling the ZP for the purpose of determining the shear modulus from the ICSI data. With regard to the NH and OGDEN models, it seems that the OGDEN model may be slightly less sensitive than the NH model. Nevertheless, it seems that there is not clear "better" model and either the NH or the OGDEN model can be used for determining the shear modulus value for the ICSI data.

[0110] It should be noted that due to the phenomena of ZP hardening shown previously (see Fig. 9) the range of aspiration length L measured in the clinical ICSI procedure drops (L=10- 18 [pm]). This is due to the fact that the applied suction pressure in the fixation stage is similar for oocytes examined before and after fertilization. For this aspiration length L range, the NH model has a slightly larger shear modulus range than the OGDEN model.EXAMPLE 5Computational parameter mapping of the oocyte fixation stage

[0111] Using oocyte specific finite element modelling is time consuming. Construction of the L vs CIO curve for one oocyte which is necessary for determining the ZP shear modulus can take many hours (18-24 hours using parallel computing on 8 cores on a typical workstation). In order to expedite the process of determining the ZP shear modulus, a computational mappingof the fixation stage parameter space was conducted. The computed aspiration length L was assumed to depend on three main factors: 1) The oocyte geometry 2) the ZP shear modulus, 3) the applied suction pressure. To take into account the geometric factor, a unit-less parameter p=t / D was defined. With t being the ZP thickness and D being the oocyte outer diameter. By taking a value of D=160 [pm] and different value of t=10-25 [pm], a series of computations on hypothetical oocytes were conducted using p=0.0625-0.15625 (the range of measured in all ICSI process of the current study was p=0.07-0.15).

[0112] Each computation was conducted for increments of C10=0.0001-0.0015 [MPa] and for a reference suction pressure of P=0.00249 [MPa]. Following the computational analysis for the different cases, all the computed data points were used to construct a 3D manifold by using thin-plate spline interpolation (MATLAB curve fitting application). Fig. 12 shows the resulting manifold in the space of p, L and CIO. The manifold shown in Fig. 12 represents only the reference suction pressure value. A separate set of computations was conducted to investigate the influence of the suction pressure P on the aspiration length L for different values of CIO. These results are shown in Fig. 13. The computations demonstrate that one can assume a linear relation between the aspiration length L and the suction pressure. This implies that for suction pressures which differ from the reference value, a linear correction factor a=P / Pref can be used to obtain the correct values from the manifold shown in Fig. 13.

[0113] In order to verify the accuracy of the parameter space mapping in determining the ZP shear modulus, the 10 oocytes previously analyzed using oocyte specific finite element modelling were used. Measured values of p and L for the 10 oocytes from the ICSI procedure were used in a MATLAB code to extract the CIO values from the manifold shown in Fig. 12. The values were corrected for the measured suction pressure of each oocyte. A comparison between the oocyte specific modelling approach and the manifold extraction method approach is shown in Fig. 14. As can be seen in Fig. 14, for most of the oocytes the CIO values obtained from the manifold extraction method are close in value to the values obtained from oocyte specific modelling. The relative difference between the two approaches is under 5% for 7 oocytes and between 10-25% for 3 oocytes before fertilization. After fertilization, the relative difference between the methods is even lower. This may suggest that the manifold shown in Fig. 12 needs to be refined in the range of higher aspiration length L values. This can be done by introducing more computation points for constructing the manifold in that specific region.EXAMPLE 6The ZP shear modulus before and after fertilization as a selection criterion

[0114] Using the manifold extraction method, discussed previously, the value of CIO was extracted for all embryos (fertilized oocyte) which were selected and transferred into the patient. In Fig. 15, the CIO values for all oocytes before fertilization are shown. In a previous study it was shown, on a different set of ICSI data, that oocytes which had CIO values in the range of 0.2-0.4 [kPa] had a greater probability of implanting into the uterus. The results in Fig. 15 further strengthen the hypothesis that the value of ZP shear modulus can be used in the embryo selection criteria to increase the potential for implantation.

[0115] The values of CIO after oocyte fertilization are shown in Fig. 16. The results in Fig. 16 show that the differences in ZP shear modulus value increase following fertilization. This is due to the fact that ZPHF (Fig. 9) is not constant. This increases the range of CIO values for which positive implantation is observed from 0.2-0.4 [kPa] to 0.2-0.6 [kPa].

[0116] In Fig. 17, the value of the ZPHF for the different transferred embryos is shown. The average ZPHF value is 1.84 with a standard deviation of 0.836 with a maximum value of 4.3. The current results are lower than ZPHF reported for animal oocyte in the literature. These studies report a value of 2.9 for Mouse oocytes and 2.69 for Porcine oocytes. Currently the results seem to indicate that the initial hypothesis of the study is correct, and that the ZP shear modulus can be used in the selection process to increase implantation potential.

[0117] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for determining suitability of an oocyte post intracytoplasmic sperm injection (ICSI) for embryo transfer, the method comprising determining at least one geometrical parameter selected from the group consisting of: zona pellucida (ZP) thickness (t), ZP outer diameter (Dout), ZP inner diameter (Din), ZP average diameter (Daverage (DOut+Din) / 2), suction length (L), any ratio thereof, and any combination thereof, wherein a value of said at least one geometrical parameter being between a predetermined range is indicative of an embryo developed from said oocyte having increased implantation probability compared to a control, thereby determining suitability of the oocyte post ICSI for embryo transfer.

2. The method of claim 1, further comprising a step of selecting said oocyte for embryo transfer.

3. The method of claim 2, wherein said selecting comprises in vitro culturing said oocyte, cryopreserving said oocyte, transferring said embryo, or any combination thereof.

4. The method of any one of claims 1 to 3, wherein said ratio is selected from the group consisting of: t / Dout, t / Din, t / Dout, t / DaVerage, L / Din, L / Dout, L / DaVerage, t / L, and any combination thereof.

5. The method of claim 4, wherein said at least one geometrical parameter, ratio thereof, or any combination thereof, is determined at a predefined pressure value.

6. The method of any one of claims 1 to 5, wherein said at least one geometrical value being below or above said predetermined range, is indicative of an embryo developed from said oocyte having equal or reduced implantation probability compared to a control.

7. The method of any one of claims 1 to 6, wherein said control comprises an oocyte the embryo developing therefrom failed to implant or did not properly implant.

8. The method of any one of claims 1 to 7, wherein any one of: said transfer, and transferring is to a receptive female.

9. The method of claim 8, wherein said female is a human female subject.

10. The method of any one of claims 1 to 9, further comprising determining a shear modulus value (Cio) of the ZP of said oocyte based on said at least one geometrical parameter, ratio thereof, and any combination thereof.

11. The method of claim 10, wherein a Cio value ranging between 0.2-0.4 is indicative of said embryo developed from said oocyte having increased implantation probability compared to said control.

12. A method for selecting an oocyte post ICSI for embryo transfer, the embryo developing therefrom having increased probability of implantation compared to a control, the method comprising determining at least one geometrical parameter selected from the group consisting of: zona pellucida (ZP) thickness (t), ZP outer diameter (Dout), ZP inner diameter (Din), ZP average diameter (DaVerage (DOut+Din) / 2), suction length (L), any ratio thereof, and any combination thereof, wherein a value said at least one geometrical parameter, ratio thereof, or any combination thereof, between a predetermined range is indicative of an embryo developing from said oocyte having increased implantation probability compared to said control, and selecting said oocyte for embryo transfer.

13. The method of claim 12, wherein said selecting comprises in vitro culturing said oocyte, cryopreserving said oocyte, transferring said embryo, or any combination thereof.

14. The method of claim 12 or 13, wherein said at least one geometrical parameter, ratio thereof, or any combination thereof, is determined at a predefined pressure value.

15. The method of any one of claims 12 to 14, wherein said control comprises an oocyte the embryo developing therefrom failed to implant or did not properly implant.

16. The method of any one of claims 12 to 15, wherein any one of: said transfer and said transferring is to a receptive female.

17. The method of claim 16, wherein said female is a human female subject.

18. The method of any one of claims 12 to 17, further comprising determining a shear modulus value (Cio) of the ZP of said oocyte based on said at least one geometrical parameter, ratio thereof, and any combination thereof.

19. The method of claim 18, wherein a Cio value ranging between 0.2-0.4 is indicative of said embryo developed from said oocyte having increased implantation probability compared to said control.

20. The method of any one of claims 1 to 19, being an in vitro or ex vivo.

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