DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election of Group I, claims 1, 10, and 75 in the reply filed on 07/17/2025 is acknowledged. New claims 86-102 fall within group I and are examined with group I. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Status of the Claims
Claims 1, 10, 75, and 86-102 are currently pending. Claims 86-102 are new; Claims 2-9, 11-74, and 76-85 are cancelled. Claims 1, 10, 75, and 86-102 are examined below.
Priority
The present application was filed as a proper National Stage (371) entry of PCT Application No. PCT/US20/17697, filed 02/11/2020, which claims benefit under 35 U.S.C. 119(e) to provisional application No. 62/803,702, filed 02/11/2019.
Information Disclosure Statement
The information disclosure statement (IDS) filed 04/20/2022 has been considered, initialed, and is attached hereto.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 92, 93 and 99 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 92 is vague and indefinite because it is not clear what reagents are included in “similar apoptotic detection reagents”. 7AAD is a DNA dye that cannot penetrate intact membranes and therefore stains dead or dying cells, whereas a cleaved caspase 3 reagent binds an enzyme involved in apoptosis. It is therefore unclear what reagent(s) would be similar to either one or both of said reagents.
Claim 93 recites “a predetermined number”. The recited language is indefinite because it is not clear how this limitation limits the method. For example, although the language appears to place a limit on the amount of the binding partners in terms of a “predetermined number”, the predetermined number is not defined, and one having ordinary skill cannot readily visualize what number is present such to clearly distinguish what is claimed from that taught by the prior art methods. Because there is no clear boundary as to the number, the claim language is indefinite.
Claim 99 recites “fluorescence-activated cell sorting (FACS)”. The recitation of FACS is indefinite because it is unclear if FACS refers to fluorescence activated cell sorting as in merely the technique, or a specific FACS® machine (i.e., employs using a fluorescence activated cell sorter by Beckton Dickinson) used to achieve the cell sorting.
Claim 99 contains the trademark/trade name FACS®. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a fluorescence activated cell sorter and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 93 is rejected under 35 U.S.C. 101 because the claimed invention is directed to law of nature/natural phenomena and an abstract idea (mental concept) without significantly more.
The U.S. Patent and Trademark Office recently revised the MPEP with regard to § 101 (see the MPEP at 2106). Regarding the MPEP at 2106, in determining what concept the claim is “directed to”, we
look to whether the claim recites:
any judicial exceptions, including certain groupings of abstract ideas (i.e., mathematical concepts, certain methods of organizing human activity such as a fundamental economic practice, or mental processes); and
additional elements that integrate the judicial exception into a practical application (see MPEP § 2106.05(a)-(c), (e)-(h)).
Only if a claim (2) recites a judicial exception and (2) does not integrate that exception into a practical application, do we then look to whether the claim contains an “’inventive concept’ sufficient to ‘transform’” the claimed judicial exception into a patent -eligible application of the judicial exception. Alice, 573, U.S. at 221 (quoting Mayo, 566 U.S. at 82). In so doing, we thus consider whether the claim:
adds a specific limitation beyond the judicial exception that is no “well-understood, routine, conventional: in the field (see MPEP § 2106.05(d)); or
simply appends well-understood, routine, conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception.
See MPEP 2106.
ELIGIBILITY STEP 2A: WHETHER A CLAIM IS DIRECTED TO A JUDICIAL EXCEPTION
Step 2A, prong 1
Claim 93 is directed to the natural correlation between the amount of living sperm cells bound to the reagent (amount of living sperm cells displaying phosphatidylserine on the cell surface) and the fertility of a sperm sample. Claim 93 recites “identifying the sample as a fertile sample if an amount of living sperm cells bound to the reagent […] exceeds a predetermined number”.
The natural relationship to which the claims are directed (i.e. between amount of living sperm cells with phosphatidylserine exposed on the cell surface and the sample being fertile) is a law of nature. Similar concepts have been held by the courts to constitute law of nature/ natural phenomena, as in the identification of a correlation between the presence of myeloperoxidase in a bodily sample (such as blood or plasma) and cardiovascular disease risk in Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1361, 123 USPQ2d 1081, 1087 (Fed. Cir. 2017). In Mayo, the Supreme Court found that a claim was directed to a natural law, where the claim required administering a drug and determining the levels of a metabolite following administration, where the level of metabolite was indicative of a need to increase or decrease the dosage of the drug. See Mayo Collaborative Services v. Prometheus Labs., Inc., 566 U.S. 66, 74 (2012).
The instant claims are similar to those as in Mayo as they involve a “relation itself [which] exists in principle apart from any human action” (id. At 77), namely the relationship between the amount of living sperm cells bound to the reagent, i.e. the amount of sperm cells with exposed phosphatidylserine on the cell surface, and the sample being fertile.
As indicated above, in addition to natural correlation, the claims are also directed to abstract ideas.
Claim 93 recites “identifying the sample as a fertile sample if an amount […] exceeds a predetermined number”.
The step of “identifying” based on comparison with a predetermined number (a cutoff/threshold value) also represents abstract ideas. Similar concepts involving comparing information regarding a sample or test subject to a control or target data have been held to be an "abstract mental process", as in University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 113 USPQ2d 1241 (Fed. Cir. 2014) which involved "comparing BRCA sequences and determining the existence of alterations", the collecting and comparing of known information in Classen, the comparing information regarding a sample or test subject to a control or target data in Ambry and Myriad CAFC, as well as Mayo (which also involved specific numerical cutoff levels).
The claimed steps of identifying a fertile sample based on the comparison to the cutoff/reference value may also be categorized as an abstract idea, namely a mental process/concept performed in the human mind, such as a practitioner simply observing the results and thinking about the quantified amount relative to the cut-off value, and making an evaluation, judgement, or opinion (See MPEP 2106). The claims, under broadest reasonable interpretation, cover performance of these steps solely within the human mind.
The recited cutoff values also constitute an abstract idea (a cutoff value being itself a mathematical concept).
Step 2A, prong 2
The above discussed step of “identifying” are insufficient themselves to integrate into a practical application because as discussed these steps themselves are directed to abstract ideas; in this case, identifying a fertile sample by comparing the amount of cells displaying phosphatidylserine on the surface to a predetermined threshold, represents the judicial exceptions and not a practical application thereof.
Regarding the independent claim (claim 1), see the claim further recites the limitations/steps “providing a sample” and “mixing the sample with a reagent”. However, these steps fail to further amount to a practical application of the indicated judicial exception(s). Specifically, the providing a sample and mixing the sample with the reagent are steps considered to be insignificant extra-solution activity, as it is a mere data gathering step (necessary in order to gather the data, namely, to count phosphatidylserine positive sperm).
ELIGIBILITY STEP 2B: WHETHER THE ADDITIONAL ELEMENTS CONTRIBUTE AN "INVENTIVE CONCEPT"
Further, the additional elements of the claims (the active method steps/limitations recited in addition to the judicial exceptions themselves) do not add significantly more to the judicial exception(s); the additional recited claim elements are recited at a high level of generality, and are not, for example limited to any specific testing technique or any non-routine/unconventional reagent.
It was routine and conventional in the assay art at the time of the invention to determine phosphatidylserine and cell viability on sperm cells using Annexin V and a membrane impermeable dye such as 7AAD or propidium iodide, see for example Wechalekar et al. (Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice. Asian journal of andrology. 2010 Jun 7;12(4):591) teaches dual fluorescent staining of spermatozoa with Annexin V-phycoerythrin and 7-AAD (Wechalekar, page 592, ‘2.4’, lines 1-2). Alternatively, Gadella et al. (Capacitation induces cyclic adenosine 3′, 5′-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells. Biology of reproduction. 2002 Jul 1;67(1):340-50) teaches staining sperm samples with annexin V and propidium iodide (Gadella, page 341, ‘Annexin V Staining’, lines 1-4). You et al. (Semen phthalate metabolites, spermatozoa apoptosis, and DNA damage: a cross-sectional study in China. Environmental science & technology. 2015 Mar 17;49(6):3805-12) similarly teaches staining sperm with Annexin V and propidium iodide (You, page 3806, ‘Annexin V Assay’, lines 11-16).
Given that assaying for phosphatidylserine and viable cells, using Annexin V and a membrane impermeable dye such as 7-AAD or propidium iodide to stain sperm cells, was routinely done in the assay art at the time, the claimed determining step fails to go beyond routine/conventional activity and fails to impose meaningful limit on the claim scope. The claimed determining step does not add any feature that is more than well-understood, purely conventional, or routine activity in the field. When recited at this high level of generality, there is no meaningful limitation, such as a particular or unconventional machine or a transformation of a particular article, in this step that distinguishes it from well-understood, routine, and conventional data gathering activity engaged in by scientists prior to applicant’s invention. See also MPEP 2106.05(g). As indicated previously above, referring to MPEP 2106.05(b), use of a machine that contributes only nominally or insignificantly to the execution of the claimed method (e.g., in a data gathering step or in a field-of use limitation) would not integrate a judicial exception or provide significantly more.
For all of these reasons, the claims fail to include additional elements that are sufficient to amount to significantly more than the judicial exception(s).
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim 75 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kaufmann et al. US7569339B2 (12/08/2016).
Regarding claim 75, Kaufmann teaches a kit comprising Annexin V and a package leaflet including information on how to use Annexin V (Kaufmann, column 3, lines 44-47). Kaufmann further teaches that Annexin V binds phosphatidylserine (Kaufmann, column 10, lines 38-39). As such Kaufmann teaches a kit comprising a reagent that selectively binds phosphatidylserine and instructional material.
Regarding the limitation “instructional material for detecting and/or isolating fertilization competent sperm cells”, MPEP 2112.01(III) states that “[w]here the only difference between a prior art product and a claimed product is printed matter that is not functionally related to the product, the content of the printed matter will not distinguish the claimed product from the prior art. In re Ngai, 367 F.3d 1336, 1339, 70 USPQ2d 1862, 1864 (Fed. Cir. 2004) (Claim at issue was a kit requiring instructions and a buffer agent. The Federal Circuit held that the claim was anticipated by a prior art reference that taught a kit that included instructions and a buffer agent, even though the content of the instructions differed, explaining "[i]f we were to adopt [applicant’s] position, anyone could continue patenting a product indefinitely provided that they add a new instruction sheet to the product."). In the instant case the limitation is not considered to contribute to the overall patentability of the claimed invention the prior art teaches a kit comprising Annexin V and information on how to use it (instructional material).
The preamble of claim 75 recites “A kit for detecting and/or isolating fertilization competent sperm cells in a sample”. Kaufmann is silent as to “detecting and/or isolating fertilization competent sperm”.
The normal purpose of a claim preamble is to recite the purpose or intended use of the claimed invention. Such statements merely define the context in which the invention operates and usually will not limit the scope of the claim (MPEP 2111.02). “[D]uring examination proceedings, claims are given their broadest reasonable interpretation consistent with the specification.” In re Hyatt, 211 F.3d 1367, 1372 (Fed. Cir. 2000). “If the claim preamble, when read in the context of the entire claim, recites limitations of the claim, or, if the claim preamble is ‘necessary to give life, meaning, and vitality’ to the claim, then the claim preamble should be construed as if in the balance of the claim.” Pitney Bowes Inc. v. Hewlett Packard Co., 182 F.3d 1298, 1305 (Fed. Cir. 1999). However, when the body of the claim fully and intrinsically sets forth the complete invention, including all of its limitations, and the preamble offers no distinct definition of any of the claimed invention’s limitations, but rather merely states, for example, the purpose or intended use of the invention, then the preamble is of no significance to claim construction because it cannot be said to constitute or explain a claim limitation. Id.
In the instant case, the statements in the preamble merely express the purpose of the claimed method and fail to clearly result in a manipulative difference. The statements in the preamble do not provide antecedent basis for terms in the body of the claim, and are not essential to understand the limitations or terms in the body of the claim.
Claim Rejections - 35 USC § 102/103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1 and 86-90 are rejected under 35 U.S.C. 102(a)(1) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Gadella et al. Capacitation induces cyclic adenosine 3′, 5′-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells. Biology of reproduction. 2002 Jul 1;67(1):340-50).
Regarding claim 1, the claim is a method of detecting fertilization competent sperm cells, the method comprising steps of providing sample, mixing sample with reagent that selectively binds PS, detecting living sperm bound to the reagent, whereby fertilization competent cells are detected. Based on the claims and the originally filed specification (which also recites on page 35, lines 21-23, that fertilization-competent sperm cells are detected by mixing a sperm cell sample with a reagent that selectively binds phosphatidylserine on the sperm cells and detecting living sperm cells bound to the reagent), detection of binding of phosphatidylserine on the surface is tantamount with determining living sperm cells that are fertilization competent. Gadella teaches that during fertilization, two membrane fusion events must take place in the sperm cell and that, unlike during spermiogenesis, sperm maturation, and storage, when the spermatozoon must remain stable, at the time of fertilization a preparatory process of membrane destabilization must take place prior to the sperm’s encountering the egg, a process known as capacitation. Gadella further teaches that the component that appears particularly responsible for inducing capacitation is bicarbonate/CO2, which acts by stimulating a special form of adenylyl cyclase leading to alterations in the lipid architecture of the plasma membrane in boar spermatozoa (Gadella, page 340, ‘Introduction’, see entire first paragraph). Gadella further teaches that the bicarbonate-induced alteration is found to be concomitant with a change in transport and transverse membrane distribution of phospholipids (Gadella, page 340, ‘Introduction’, 2nd paragraph, lines 1-7), resulting in exposure of phosphatidylserine at the outer surface (Gadella, page 341, lines 7-10). Put another way, Gadella teaches that sperm cells go through capacitation in order to become fertilization competent and this results in the exposure of phosphatidylserine at the outer surface of the sperm cell. Gadella further teaches that annexin V specifically labels surface-exposed phosphatidylserine (reagent that specifically binds; Gadella, page 341, 2nd paragraph, lines 11-12). Gadella further teaches taking sperm samples (providing a sample) and labeling them with annexin V-FL (mixing the sample with a reagent that binds phosphatidylserine) and propidium iodide to discriminate between live and dead cells (Gadella, page 341, ‘Annexin V Staining’, lines 1-3). Gadella further teaches that staining spermatozoa with annexin V and propidium iodide showed that bicarbonate induced exposure of phosphatidyl serine in a substantial subpopulation of intact cells (detect fertilization competent cells; Gadella, page 343, 2nd column, lines 4-6).
Put another way, Gadella teaches that labelling sperm induced by bicarbonate to be in a fusible state (fertilization competent) with Annexin V and propidium iodide detects living cells that demonstrate a change in transport and transverse membrane distribution of phospholipids as shown by the membrane exposure of phosphatidylserine and are therefore fertilization-competent. As such Gadella teaches a method of detecting fertilization competent sperm cells and therefore anticipates claim 1.
Although Gadella is considered to anticipate the claimed invention, because they are correlating binding with functional/living sperm, and as a result, fertilization competent sperm, it is also acknowledged that Gadella does not explicitly indicate that they are indicating these sperm as “fertilization competent” (see Gadella uses the language that these sperm have undergone capacitation) and as such, the present rejection under 35 U.S.C. 103 is also being made. Additionally to the reasoning provided above (under 35 U.S.C. 102), it would have also been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have used the method of Gadella (of staining boar sperm cells with Annexin V and propidium iodide to detect fertilization-competent sperm cells) to specifically indicate the cells are fertilization competent because of the teaching of Gadella that sperm that has undergone capacitation (a process of membrane destabilization of the sperm cell necessary to prepare for fertilization), is detectable by Annexin V labeling of phosphatidylserine which is exposed on the surface of live cells.
Regarding claims 86 and 87, Gadella teaches boar spermatozoa (non-human animal; porcine; Gadella page 340, ‘Abstract’, line 6).
Regarding claim 88, Gadella teaches that sperm-rich fraction of boar semen were collected, diluted and stored. For experimentation, spermatozoa were isolated from the sperm-rich fractions by centrifugation and the resultant loose pellet was resuspended (Gadella, page 341, see ‘Sperm Cell Preparation’). Put another way, Gadella teaches concentrating spermatozoa from boar semen into a pellet and therefore teaches concentrating the semen sample.
Regarding claim 89-90, Gadella teaches taking sperm samples and labeling them with annexin V-FL and propidium iodide to discriminate between live and dead cells (Gadella, page 341, ‘Annexin V Staining’, lines 1-3). Gadella further teaches Annexin V-FL is fluorescein-conjugated annexin V, which specifically detects phosphatidylserine (Gadella, page 341, 3rd paragraph, lines 4-6).
Claim 91 is rejected under 35 U.S.C. 103 as being unpatentable over Gadella et al. as applied to claim 1 above and further in view of Koç et al. The detailed comparison of cell death detected by annexin V-PI counterstain using fluorescence microscope, flow cytometry and automated cell counter in mammalian and microalgae cells. Journal of fluorescence. 2018 Nov;28(6):1393-404 and McPherson et al. Semen effects on insemination outcomes in sows. Animal reproduction science. 2014 Dec 10;151(1-2):28-33.
Regarding claim 91, Gadella teaches taking sperm samples and labeling them with annexin V-FL and propidium iodide to discriminate between phosphatidylserine positive live and dead cells (Gadella, page 341, ‘Annexin V Staining’, lines 1-3). Gadella further teaches percentage of intact phosphatidylserine expressing cells by flow cytometry (Gadella, page 343, 2nd column see 2nd paragraph, and figure 2B). Gadella teaches using flow cytometric analysis of sperm cells comprising detecting Annexin V-FL fluorescence on FL-1 and PI fluorescence on FL-3. Gadella further teaches setting up 2-dimensional dot plots with FL-1 (Annexin V labeled live cells) on one axis and FL-3 (PI stained dead cells) on another in order to exclude PI stained cells and calculating the relative proportions of the Annexin V-negative and the Annexin V-positive populations.
Gadella does not teach counting a total number of sperm cells, counting apoptotic and/or necrotic sperm cells in the sample, and counting sperm cells bound to the reagent minus the apoptotic and/or dear sperm cells.
Koç teaches the evaluation of mammalian cell wellness by annexin V-Propidium iodide counterstaining to discriminate apoptotic and necrotic cell profiles. Koç teaches a fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter (Koç, Title and ‘Abstract’, lines 1-5). Koç teaches evaluating the total cell number (Figure 1 e) and live and dead cells in a mammalian cell line MDA-MBD213 by automated cell counter (Figure 1 b and d; Koç, page 1395, see Figure 1). Koç further teaches the same evaluation in a second mammalian cell line AR42J (Koç, page 1396, see Figure 2). Koç further teaches analyzing Annexin V and PI staining using an automated cell counter (Koç, page 1396, 2nd paragraph, lines 3-4). As such Koç teaches counting a total number of cells in a sample, counting necrotic cells and counting cells bound to Annexin V.
McPherson teaches that artificial insemination is common practice in the global pig industry with reliance on extended, chilled semen permitting more rapid genetic gain than that achievable via natural breeding. This places more emphasis on quantitative and qualitative aspects of boar semen. McPherson further teaches that when sperm numbers per inseminate are reduced to increase the number of artificial insemination doses per ejaculate, compensable defects become more important as these can lead to reduced litter size and thus, failure to monitor boar semen quality can result in significant economic loss, especially when one problem boar is used to inseminate large numbers of females (McPherson, page 28, ‘Introduction’, see entire first paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Gadella of determining the percentage of live cells expressing phosphatidylserine on the cells surface with the method of Koç of counting cells labeled with Annexin V (expressing phosphatidylserine) and stained with propidium iodide (dead cells) in the total cell population because of the teaching of Gadella that live cells expressing phosphatidylserine (Annexin V-positive, PI-negative) are fertilization competent and the teaching of McPherson that the quality and quantity of sperm is important in artificial insemination in pigs because as the sperm numbers per inseminate are reduced, defects in sperm become more important as these can lead to reduced litter sizes. One of ordinary skill in the art would be motivated to do so because failure to monitor boar semen quality can result in significant economic loss, especially when one problem boar is used to inseminate large numbers of females.
One of ordinary skill in the art would have a reasonable expectation of success in doing so because Koç teaches counting mammalian cells stained with Annexin V and propidium iodide using laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter and Gadella shows success identifying different populations of boar semen using Annexin V and propidium iodide staining and a flow cytometer. Put another way, Gadella shows success detecting Annexin V and propidium iodide stained cells using a laser beam-employed instrument, namely a flow cytometer, and therefore one of ordinary skill in the art would reasonably expect another laser beam-employed instrument, an automated cell counter, to also be able to detect Annexin V and propidium iodide stained cells.
Claim 92 is rejected under 35 U.S.C. 103 as being unpatentable over Gadella et al. in view of Koç et al. and McPherson et al. as applied to claim 91 above and further in view of Jiang et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nature protocols. 2016 Apr;11(4):655-63.
Regarding claim 92, Gadella teaches a method of detecting fertilization competent sperm substantially as claimed.
Gadella teaches taking sperm samples and labeling them with Annexin V-FL and propidium iodide to discriminate between phosphatidylserine positive live and dead cells (Gadella, page 341, ‘Annexin V Staining’, lines 1-3).
Gadella does not teach that counting apoptotic and/or necrotic cells comprises staining the sample with 7AAD.
Jiang teaches that the use of Annexin V and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard (Jiang, page 655, Abstract, lines 1-2). Jiang further teaches that the Annexin V binding and PI/7-AAD uptake assay and subsequent data analysis is very effective in determining cell viability (Jiang, page 655, 2nd column, 2nd paragraph, lines 1-3). Jian further teaches that propidium iodide is a membrane-impermeable nucleic acid-binding dye that can be replaced by another membrane-impermeable nucleic acid-binding dye, 7AAD (Jiang, page 655, ‘Introduction’, lines 21-22 and 27-28).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the invention of Gadella in order to use 7AAD to label dead cells in place of propidium iodide as taught by Gadella as an obvious matter of a simple substitution of one art recognized membrane impermeable nucleic acid binding dye over another, both recognized as suitable for the same purpose, both effective in determining cell viability. One of ordinary skill in the art would be motivated to do so, because the propidium iodide of Gadella performs the same function specified in the claim in substantially the same way and produces substantially the same results of labeling dead and dying cells. Substituting one membrane-impermeable nucleic acid staining dye such as propidium iodide of Gadella with a different membrane-impermeable nucleic acid staining dye both of which have been successfully used in cell viability assays would yield a predictable result.
One of ordinary skill in the art would have a reasonable expectation of success in doing so because of the teaching of Jiang that an assay comprising propidium iodide or 7AAD together with Annexin V is very effective in determining cell viability.
Claim 93 is rejected under 35 U.S.C. 103 as being unpatentable over Gadella et al. as applied to claim 1 above and further in view of Martinez et al. Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows. Reproduction. 2002 Jan 1;123(1):163-70, Koç et al. and McPherson et al.
Regarding claim 93, Gadella teaches a method of detecting fertilization competent sperm by detecting phosphatidylserine positive live sperm cells substantially as claimed.
Gadella does not teach identifying a fertile sample as a sample if an amount of living sperm cells bound to the reagent exceeds a predetermined number.
As discussed previously in detail above, Koç teaches the evaluation of mammalian cell wellness by annexin V-Propidium iodide counterstaining to discriminate apoptotic and necrotic cell profiles. Koç teaches a fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter (Koç, Title and ‘Abstract’, lines 1-5). Koç teaches evaluating the total cell number (Figure 1 e) and live and dead cells in a mammalian cell line MDA-MBD213 by automated cell counter (Figure 1 b and d; Koç, page 1395, see Figure 1). Koç further teaches the same evaluation in a second mammalian cell line AR42J (Koç, page 1396, see Figure 2). Koç further teaches analyzing Annexin V and PI staining using an automated cell counter (Koç, page 1396, 2nd paragraph, lines 3-4). As such Koç teaches counting a total number of cells in a sample, counting necrotic cells and counting cells bound to Annexin V.
Martinez teaches that there is a minimum number of spermatozoa required for normal fertility in sows (Martinez, see title). Martinez further teaches that deep intrauterine insemination was performed with 15.0, 5.0, 2.5, and 1.0 x 107 spermatozoa and that there was no difference in farrowing rates after insemination with 15 or 5 x 107 spermatozoa but a significant decrease was observed in sows inseminated with 2.5 or 1 x 107 (Martinez, page 163, Abstract, lines 11-25). Martinez further teaches that sperm was derived from boars of proven fertility and with satisfactory semen characteristics (Martinez, page 164, ‘Deep intrauterine inseminations’, lines 1-2). Martinez teaches that insemination with 15.0 and 5. 0 x 107 spermatozoa results in 86% and 78% pregnancy rates respectively but insemination with 2.5 x 107 spermatozoa only results in a 52% pregnancy rate and 1.0 x107spermatozoa only result in a 39% pregnancy rate (Martinez, page 166, see table 1).
McPherson teaches that artificial insemination is common practice in the global pig industry which places more emphasis on quantitative and qualitative aspects of boar semen. McPherson further teaches that when sperm numbers per inseminate are reduced to increase the number of artificial insemination doses per ejaculate, compensable defects become more important as these can lead to reduced litter size and this can result in significant economic loss, especially when one problem boar is used to inseminate large numbers of females (McPherson, page 28, ‘Introduction’, see entire first paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Gadella of determining the percentage of life cells expressing phosphatidylserine on the cells surface with the method of Koç of counting cells labeled with Annexin V (expressing phosphatidylserine) and stained with PI (dead cells) in the total cell population because of the teaching of Gadella that cells live cells expressing phosphatidylserine (Annexin V-positive, PI-negative) are fertilization competent and the teaching of Martinez that there is a minimum number of sperm cells required for normal fertility in pigs and that farrowing rates after insemination with a number of sperm cells below the threshold show a significant decrease. One of ordinary skill in the art would be motivated to have modified the method of Gadella with the method of Koç of counting the fertilization competent sperm in a sample to ensure the amount of living sperm cells exceed the threshold because of the teaching of McPherson that the quality and quantity of sperm is important in artificial insemination in pigs because as the sperm numbers per inseminate are reduced defects in sperm can lead to reduced litter sizes and result in significant economic loss, especially when one problem boar is used to inseminate large numbers of females.
One of ordinary skill in the art would have a reasonable expectation of success having modified applying the method of Gadella and the prior art to identify a sample as a fertile sample because Gadella teaches success labelling live fertilization competent spermatozoa with annexin V and propidium iodide and Koç similarly uses annexin V and propidium iodide to determine cell numbers and Martinez teaches using a certain number of spermatozoa to achieve high pregnancy rates.
Claims 10, 94-101 are rejected under 35 U.S.C. 103 as being unpatentable over Gadella et al. in view of Paasch et al. Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. Journal of andrology. 2003 Mar 4;24(2):246-52. and McPherson et al.
Regarding claims 10 and 100-101, similarly as discussed above regarding claim 1, the claims and the specification of the present application recites on page 35, lines 21-23, that fertilization-competent sperm cells are detected by mixing a sperm cell sample with a reagent that selectively binds phosphatidylserine on the sperm cells and detecting living sperm cells bound to the reagent, i.e., detection of binding of phosphatidylserine on the surface is tantamount with determining living sperm cells that are fertilization competent.
As explained previously in detail above, Gadella teaches detecting fertilization competent sperm samples by taking sperm samples (providing a sample) and labeling said sperm samples with annexin V-FL (mixing the sample with a reagent that binds phosphatidylserine) and propidium iodide (Gadella, page 341, ‘Annexin V Staining’, lines 1-3).
Gadella does not teach a step of isolating fertilization competent sperm (claim 10).
Paasch teaches a technique for separating spermatozoa by magnetic-activated cell sorting after binding superparamagnetic annexin V-conjugated microbeads to membrane phosphatidylserine (Paasch, page 246, ‘Abstract, lines 1-4). Paasch teaches dividing sperm (human) suspensions into two fractions using a magnetic field by incubating spermatozoa with annexin V-conjugated microbeads (capture moiety/microbead) and the loading the spermatozoa/microbeads suspension on a separation column containing iron balls. The fraction composed of phosphatidylserine-positive membranes spermatozoa (annexin V-conjugated microbead+) was retained in the separation column (isolating the capture moiety) and after removing the column from the magnetic field the retained fraction was eluted using annexin V-binding buffer (Paasch, page 247, see entire paragraph ‘Depletion of Spermatozoa With Deteriorated Membrane by Magnetic Cell Separation’). Paasch further teaches that annexin V-conjugated microbeads are useful, specific, and sensitive method (Paasch, page 250, 2nd paragraph, lines 1-2).
McPherson teaches that artificial insemination is common practice in the global pig industry with reliance on extended, chilled semen permitting more rapid genetic gain than that achievable via natural breeding. This places more emphasis on quantitative and qualitative aspects of boar semen. McPherson further teaches that when sperm numbers per inseminate are reduced to increase the number of artificial insemination doses per ejaculate, compensable defects become more important as these can lead to reduced litter size and thus, failure to monitor boar semen quality can result in significant economic loss (McPherson, page 28, ‘Introduction’, see entire first paragraph).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Gadella of identifying fertilization competent sperm by staining for Annexin V and propidium iodide with the method of isolating fertilization competent sperm using magnetic Annexin V covered beads in order to magnetically sort and isolate the cells determined to be fertilization competent as taught by Paasch because of the teaching of Paasch that magnetic sorting comprising Annexin V coated beads is useful, specific and sensitive in separating the sperm into two fractions. One of ordinary skill in the art would have been motivated to have used the method of Paasch for separating human sperm to have modified the method of Gadella in order to separate boar sperm into phosphatidylserine positive and phosphatidylserine negative fractions because of the teaching of Gadella that phosphatidylserine positive live sperm cells are fertilization competent. The ordinary artisan would have been further motivated to do so because of the teaching of McPherson that in artificial insemination in pigs when sperm numbers per inseminate are reduced to increase number of artificial insemination doses per ejaculate compensable defects in the spermatozoa can lead to reduced litter size and economic loss.
Put another way, one of ordinary skill in the art would be motivated to select, i.e., isolate, fertilization competent sperm for artificial insemination in pigs in order to avoid small litter sizes when the overall number of sperm cells per inseminate are reduced to avoid economic loss.
One of ordinary skill in the art would have a reasonable expectation of success in having modified the method of Gadella of identifying fertilization competent sperm by staining for Annexin V and propidium iodide with the method of Paasch of isolating sperm using Annexin V coated microbeads because Gadella teaches success labeling phosphatidylserine expressing spermatozoa with Annexin V and the separation method as taught by Paasch similarly relies on specific binding of phosphatidylserine expressing spermatozoa to Annexin V. As such one of ordinary skill in the art would have reasonably expected that the phosphatidylserine on boar spermatozoa would be bound by microbeads coated with Annexin V.
Regarding claims 94 and 95, Gadella teaches boar spermatozoa (non-human animal; porcine; Gadella page 340, ‘Abstract’, line 6).
Regarding claim 96, Gadella teaches that sperm-rich fraction of boar semen were collected, diluted and stored. For experimentation, spermatozoa were isolated from the sperm-rich fractions by centrifugation and the resultant loose pellet was resuspended (Gadella, page 341, see ‘Sperm Cell Preparation’). Put another way, Gadella teaches concentrating spermatozoa from boar semen and therefore concentrating the semen sample.
Regarding claim 97-98, Gadella teaches taking sperm samples and labeling them with annexin V-FL and propidium iodide to discriminate between live and dead cells (Gadella, page 341, ‘Annexin V Staining’, lines 1-3). Gadella further teaches Annexin V-FL is fluorescein-conjugated annexin V, which specifically detects phosphatidylserine (Gadella, page 341, 3rd paragraph, lines 4-6).
Regarding claim 99¸ Gadella and the cited art above teaches a method of isolating fertilization competent sperm substantially as claimed.
Gadella and the cited art above do not teach that isolating sperm cells comprises employing flow cytometry or fluorescence activated cell sorting.
As explained previously in detail above, Paasch teaches separating annexin V-positive and annexin V-negative populations of sperm cells using magnetic activated cells sorting.
Paasch further teaches evaluating the system using flow cytometric analysis with anti-annexin V-FITC antibodies (Paasch, page 247, ‘Evaluation of ANMBs on Spermatozoa’, lines 1-6). Paasch further teaches that the flow cytometric analyses confirmed the separation effect and determined that the system was 95% specific and 73% sensitive (Paasch, page 249, 3rd paragraph, lines 6-8).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Gadella and the cited art above of isolating fertilization competent sperm with the method of Paasch of analyzing the separate populations for Annexin V staining in order to confirm the separation. One of ordinary skill in the art would have been motivated to do so in order to confirm the specificity and sensitivity of the system.
The ordinary artisan would have a reasonable expectation of success because Gadella teaches success using flow cytometry to detect Annexin V binding in order to evaluate the sperm quality.
Claim 102 is rejected under 35 U.S.C. 103 as being unpatentable over Gadella et al. in view of Paasch et al. Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. Journal of andrology. 2003 Mar 4;24(2):246-52. and McPherson et al. and further in view of Biocompare Isolate specific cell populations with magnetic separation systems; 05/30/2013 accessed 12/13/2025 and Vogt et al. Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation. American journal of obstetrics and gynecology. 1997 Oct 1;177(4):964-72.
Regarding claim 102¸ Gadella and the cited art above teaches a method of isolating fertilization competent sperm substantially as claimed.
Gadella does not teach eluting sperm cells form the reagent that selectively binds phosphatidylserine on the sperm cells.
Biocompare teaches that working with magnetic separation system positive selection is the simplest approach but that the downside is that the cells are stuck to antibodies which are stuck to magnetic beads and that there is concern that the bead-cell attachment could interfere with downstream experiments, for example if examining gene expression, the binding of a cell-surface receptor to an antibody might alter the transcriptome (Biocompare, ‘Positive selection’, lines 1, 5-6, and 12-14).
Vogt teaches using BeWo choriocarcinoma cells (Vogt, page 965, 2nd paragraph, line 2) and further teaches staining BeWo cells with fluorescently labeled annexin V (Vogt, page 965, see ‘Fluorescein isothiocyanate-annexin-V binding’). Vogt further teaches that adding anti-phosphatidylserine antibody removed most annexin-V from the BeWo surface (Vogt, page 965, lines 1-4).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Gadella and the cited art above of isolating fertilization competent sperm with the method of Vogt of adding anti-phosphatidylserine antibodies in order to release Annexin V binding to phosphatidylserine as taught by Vogt in order to release the beads. One of ordinary skill in the art would be motivated to do so because of the teaching of Biocompare that there is concern that the attachment could interfere with downstream experiments.
One of ordinary skill in the art would have a reasonable expectation of success in removing the microbeads by adding anti-phosphatidylserine antibodies, because of the teaching of Vogt of having removed Annexin V bound to a sample with the same method.
Conclusion
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677