WO2012005606A1 - Methods and kits for infertility testing - Google Patents
Methods and kits for infertility testing Download PDFInfo
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- WO2012005606A1 WO2012005606A1 PCT/NZ2011/000122 NZ2011000122W WO2012005606A1 WO 2012005606 A1 WO2012005606 A1 WO 2012005606A1 NZ 2011000122 W NZ2011000122 W NZ 2011000122W WO 2012005606 A1 WO2012005606 A1 WO 2012005606A1
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- sprasa
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Definitions
- the invention generally relates to methods and kits for identifying the cause of infertility and furthermore identifying the likelihood of an infertile subject responding to infertility treatment.
- Infertility relates to the biological inability to conceive after 12 months of regular unprotected intercourse.
- the World Health Organisation has identified infertility as a public health issue and there is a need for more research in this area.
- ASA antisperm antibodies
- IVF in vitro fertilisation
- SPRASA Antisperm Antibodies
- SLLP-1 and SPACA-3 antibodies reactive with Sperm Protein Reactive with Antisperm Antibodies
- SLLP-1 and SPACA-3 antibodies reactive with Sperm Protein Reactive with Antisperm Antibodies
- SLLP-1 and SPACA-3 antibodies reactive with Sperm Protein Reactive with Antisperm Antibodies
- SPRASA is expressed by oocytes and maybe expressed by preimplantation embryos (Wagner, A., et al, 2006; Wagner, A. et al, 2008).
- SPRASA protein There are at least two iso forms of the SPRASA protein, the long iso form of the protein is encoded by exons 1-5 of the gene, while a short iso form is encoded only by exons 2-5.
- the long iso form appears to be expressed only in sperm of higher primates while the short isoform is more widely expressed and is present in the sperm and oocytes of many mammalian species (Prendergast, Woad et al. 2008; Prendergast, Woad et al. 2008).
- Expression of SPRASA by both oocytes and sperm suggests that this protein has an important function in fertility.
- SPRASA antibodies there is no information on the incidence of SPRASA antibodies in humans, and whether there is a correlation between SPRASA-reactive antibodies and infertility.
- the present invention relates broadly to a prognostic screen of women seeking fertility treatment to exclude those who have SPRASA reactive antibodies (and who would be expected to respond less well to some infertility procedures/treatment than others).
- the present invention provides a method of identifying the likelihood of an infertile subject responding to infertility treatment comprising:
- the present invention provides a method of diagnosing cause of infertility in a subject comprising:
- a method of determining the predisposition of a subject to developing a pregnancy pathology comprising
- Pregnancy pathologies can include but are not limited to foetal death, recurrent miscarriage, uterine adhesions, abnormalities of the uterus and the like.
- the subject is a human and even more preferably the subject is a female human subject.
- the methods of the present invention are equally useful and applicable to other species.
- the body fluid collected from the infertile subject is blood, which may be processed to plasma or serum, or may include follicular fluid, cervical mucus, semen or seminal plasma or uterine washings.
- the SPRASA reactive antibody to be measured and/or detected is IgG but IgA or IgM antibodies may also be detected/measured using the same methods and assay formats.
- the antibody to be detected in the body fluid sample may either be a free antibody or it may be complexed to the SPRASA antigen.
- the SPRASA reactive antibody to be measured and/or detected is IgG but IgA or IgM antibodies may also be detected/measured using the same methods and assay formats.
- the antibody to be detected in the body fluid sample may either be a free antibody or it may be complexed to the SPRASA antigen.
- the SPRASA reactive antibody to be measured and/or detected is IgG but IgA or IgM antibodies may also be detected/measured using the same methods and assay formats.
- the antibody to be detected in the body fluid sample may either be a free antibody or it may be complexed to the SPRASA antigen.
- the SPRASA reactive antibody to be measured and/or detected is IgG but Ig
- control level of antibody may be set by measurements of antibody levels in blood of a population of fertile subjects. However, it will be understood that control levels of antibody may be determined in any other suitable body fluid sample and population of subjects, depending on requirements.
- IVF in vitro fertilisation
- ICSI intracytoplasmic sperm injection
- IVF in vitro fertilisation
- ICSI intracytoplasmic sperm injection
- hormone treatment hormone treatment and the like.
- kits for detecting and/or measuring anti-SPRASA antibodies in a fluid sample comprising SPRASA antigen or a functional part thereof and reagents for detection of anti-SPRASA antibodies or anti- SPRASA antibody/SPRASA complex.
- test kits of the present invention may include a porous surface or solid substrate to which the antigen has been preabsorbed or covalently bound, said surface substrate being preferably in the form of microtitre plates or wells; test sera; various diluents and buffers; labelled conjugates for the detection of specifically bound antibodies and other signal-generating reagents such as enzyme substrates, co factors and chromogens.
- the SPRASA antigen or protein, or a functional part thereof can be obtained by recombinant or synthetic means.
- recombinant SPRASA product can span individual or multiple exons of the SPRASA coding sequence. It will be understood that fragments, motifs and mimetics of SPRASA that have functional or specific antigenic activity can also be used.
- antibody/SPRASA complex may be selected from those conventionally used in the Enzyme-linked immunosorbent assay (ELISA) format, for example labelled (e.g.
- heterologous species anti-IgG, IgA or IgM antibodies can be biotinylated antibodies anti-IgG, IgA or IgM antibodies.
- a radionuclide e.g. 131 I, 3 H or similar.
- detection antibodies may be labelled with a varity of fluorophores for example, fluorescene isothiocyanate (FITC).
- FITC fluorescene isothiocyanate
- Figure 1 Photograph of purified recombinant SPRASA (exons 2-5, short isoform) band at 15kDa .
- Figure 2 Photograph of purified recombinant SPRASA(exonsl -5, long isoform) band at 26 kDa.
- Figure 3 IgG immunoreactivity of infertile and fertile women against recombinant SPRASA exons 2-5 (short isoform). Infertile patients M27, 45 and 48 have higher levels of SPRASA reactive IgG antibodies than other infertile and fertile women.
- Figure 4 IgG immunoreactivity of sera from men and women in the fertile and infertile groups against recombinant SPRASA exons 1-5 (long isoform). Horizontal line indicates the cut-off level for high anti-SPRASA antibody levels. (0.8 is the value for the cut-off level as determined by normalising values of the test samples against the values obtained for corresponding reference samples included in each ELISA performed. The test samples are expressed as a percentage of the median of the reference samples.) - 13 infertile individuals and 1 fertile individual are shown to have anti-SPRASA antibody levels above the cut-off. Of the infertile individuals 6 were men and 7 were women. 4 of these individuals had otherwise unexplained infertility.
- Figure 5 Mean uterine weights of females immunised with SPARS A and keyhole limpet peptide (KLH), respectively, and mated correspondingly with males immunised subcutaneously (s.c.) with SPRASA and KLH, respectively, at 12 days post- coitus (dpc). Data are expressed as mean + standard deviation (SD). There were no significant differences in the uterine weights between SPRASA and KLH immunised females.
- SD standard deviation
- Figure 6 Mean litter size of females immunised with SPARSA and KLH, respectively, and mated correspondingly with males immunised s.c. with SPRASA and KLH, respectively, at 12 dpc. Data are expressed as mean + SD. There were no significant differences in the litter size between SPRASA and KLH immunised females.
- Figure 7 Mean individual pup weights of females immunised with SPARSA and KLH, respectively, and mated correspondingly with males immunised (s.c.) with SPRASA and KLH, respectively, at 12 dpc. Data are expressed as mean + SD. There were no significant differences in the individual pup weight between SPRASA and KLH immunised females.
- Figure 8 Representative photographs of uteri from (A) SPRASA and (B) KLH immunised females at 12 dpc. Uteri from SPRASA immunised females appeared distinctly different from the uteri from KLH immunised females. Embryo clustering and reabsorptions seen in SPRASA immunised females are indicated by black and white arrows, respectively.
- Figure 10 Mean litter size of 10 Wistar rats immunised against recombinant
- SPRASA short isoform; exons 2-5) were significantly ( p 0.0268) smaller than the mean litters size of control Wistar rats immunised against KLH. Both groups of immunised female were mated with unimmunised proven stud males.
- Figure 1 1 Shows the mRNA nucleotide sequence of exons 1 to 5 of the mouse (A; gi
- Exons are indicated by alternating underlined and bold nucleotides (i.e. in Figure 1 A, the sequence of exon 1 is underlined, the sequence of exon 2 is in bold text, the sequence of exon 3 is underlined again, and so forth); the start ATG codon is indicated by a white box.
- Figure 12 Shows the mRNA nucleotide sequence of exons 1 to 5 of the human SPRASA gene (gi
- Figure 13 Shows an alignment of the human, mouse and rat SPRASA exon sequences.
- the start ATG codon is indicated as bold and underlined nucleotides.
- the present invention is based in part on the studies which demonstrate that not only is SPRASA expressed by oocytes, where it is present on the oolemmal membrane and in the zona pellucida, but that some infertile subjects have a circulating antibody reactive with SPRASA.
- Antisperm (and anti-oocyte) antibodies have long been known to be associated with infertility but these antibodies may also be found in fertile persons. This limits somewhat the diagnostic/prognostic value of antisperm antibody testing. This is in part because current tests do not distinguish between sperm/ovarian antigens that are crucial for fertility and those that are not.
- mice immunised with SPRASA were profoundly infertile while female rats immunised with SPRASA (exons 2-5; short isoform) had significantly smaller mean litter sizes than control immunised female rats (Figure 10). This suggests that, in affected couples, SPRASA antibodies may be a dominant cause of their infertility.
- the methods and kits of the present invention are simple and inexpensive diagnostic and prognostic tools, effective in identifying causes of infertility in women, informing treatment decisions or at least informing possible lack of response to conventional infertility treatments.
- An added advantage of the methods and kits of the present invention is that certain pregnancy complications and pathologies (including miscarriage and still birth) can be predicted should fertilisation take place, based on measurement of circulating anti-SPRASA antibody or anti-SPRASA antibody/SPRASA complex.
- PCR products (Forward primer: aaagtggatccatgttgttggccctggtctgt and Reverse primer: cccgggaattctca gaagtcacagccatccaccca) were cloned into a protein expression vector (pPROX HTb), and the recombinant plasmids were transformed in E.coli, and sequenced to confirm the cloned products. Single colonies of E.coli expressing the recombinant plasmids were grown at 37°C in an overnight culture of 10ml Luria broth (LB) containing 100 ⁇ g/ml ampicillin, and used to inoculate 2L of pre-warmed LB media.
- LB Luria broth
- Recombinant SPRASA expression was induced with the addition of ImM Isopropyl-P-D-thiogalactopyranoside (IPTG) and the culture was incubated overnight at 37°C.
- Cells were lysed by a cell disruptor at 18 ksi, and the soluble and insoluble fractions separated by centrifugation at 15,000 x g for 20 minutes. The supernatant was discarded, and the insoluble pellet was dissolved in Buffer A with shaking on a rotator. Supernatant from the insoluble fraction was then filtered and loaded onto a 5ml Ni- charged Hi-Trap chelating column. The column was washed with 20ml of Buffer B
- the purity of eluted Histidine-tagged recombinant SPRASA was evaluated by Coomassie Blue staining following SDS PAGE, and by western blotting. 20 ⁇ from each of the 2ml fractions were mixed with equal volume of 2x sample buffer and separated on a 15% polyacrylamide gel in a Mini-Protean II Cell at 120V under reducing conditions (10% 2-mercaptoethanol). Protein bands were then stained with Coomassie Brilliant Blue G250 to detect a band at the predicted molecular weight (approximately 15 kDa) or transferred to a 0.45 ⁇ nitrocellulose membrane (Hybond-C extra,
- Membranes were then washed three times in PBS-Tween and incubated for an hour with horseradish peroxidase (HRP) conjugated streptavidin (Zymed Laboratories, USA) at 1 :5000 dilution in blocking solution. After three further PBS-Tween washes, the signals were detected by enhanced chemiluminescence (Amersham, Auckland), with 1 minute X-ray film exposure time, followed by processing in an automated film developer (Agfa Curix60, Germany).
- HRP horseradish peroxidase
- Ni-affinity chromatography fractions containing SPRASA were pooled and separated on a 15% polyacrylamide gel as described previously. Two thin strips were excised from either side of the gel and stained with Coomassie Blue to identify protein bands at the predicted molecular weight of recombinant SPRASA (15kDa). Following staining, the strips were destained in destaining solution, and rehydrated in tap water. The strips were then used as a guide for excising the protein band at 15kDa on the unstained gel. The excised gel strip was minced and left overnight at 4°C in 8M urea in cold PBS.
- EXAMPLE 2B Expression and purification of Histidine-tagged Recombinant Human SPRASA (long isoform; exons 1-5)
- a recombinant SPRASA gene (exons 1-5) lacking the trans-membrane region (amino acids MLLALVCLLSCLLPSSEA of wild-type SPRASA) and having an N- terminal hexa-histidine sequence (optimised for codon usage in E. coli) was produced by GenScript of Wheelock House, Level 18, 20 Pedder Street, Central, Hong Kong.
- the construct was cloned into the plasmid pUC57.
- the DNA sequence of the SPRASA construct was:
- This expression construct encodes for a protein having the following amino acid sequence:
- This protein was expressed in E. coli and purified from inclusion bodies by nickel chlorine affinity chromatography as described for histidine-tagged recombinant short SPRASA isoform (exons 2-5; see Example 2A above). After solubilisation of the inclusion bodies the protein was resuspended in phosphate buffered saline containing 0.2% sodium lauroyl sarcosine, pH 8.0.
- Fertile and infertile patients recruited for this investigation were accessed through the maternity services of National Women's Hospital following radio advertisements, and Fertility Plus (the fertility service of Auckland District Health Board, NZ), respectively, as well as through Fertility Associates. Blood samples were allowed to clot overnight at 4°C and processed the following morning by centrifugation, at 2400 x g for 10 minutes. Serum was aspirated, aliquoted and stored at -80°C until required.
- Non-specific binding sites were blocked with 40 ⁇ 1 of 5% non-fat milk powder in PBS-Tween for an hour at room temperature. After three washes with PBS-Tween, the plates were incubated with patient sera at 1 : 100 dilution in blocking solution for an hour at room temperature. The plates were then washed three times in PBS-Tween, and incubated with biotinylated mouse anti-human IgG or IgA, diluted 1 :2000 in blocking solution for an hour at room temperature.
- HRP horseradish peroxidase
- streptavidin 1 :2000 (for IgG assay) orl :5000 (for IgA assay) in blocking solution for an hour, followed by three more washes in PBS-Tween.
- HRP horseradish peroxidase
- POD o-phenylenediamine dihydrochloride
- Control mice were immunised with keyhole limpet peptide (KLH).
- KLH keyhole limpet peptide
- Each animal received at least three immunisations at two week intervals, and each immunisation consisted of 50uL recombinant SPRASA protein (25 ⁇ g) or KLH peptide (25 ⁇ g) in PBS, emulsified with 50 ⁇ , of adjuvant.
- the first immunisation was given with Freund's Complete adjuvant, and subsequent booster immunisations employed Freund's Incomplete adjuvant.
- Each animal was immunised four times at two weekly intervals, with ⁇ . aluminium hydroxide suspension containing 5 ⁇ g of recombinant SPRASA or KLH.
- Ovarian tissue from SPRASA and control immunised female mice in dioestrus were excised and fixed in 4% PFA overnight at 4°C. The following morning, ovaries were dehydrated in 70% ethanol at room temperature, cleared in xylene (BDH,
- Follicle populations in the ovaries of SPRASA and control KLH immunised females were evaluated using light microscopy.
- the follicle classification system employed was described previously by Myer et al., 2004:
- primordial follicles are characterised by oocytes surrounded by a flattened
- Primary follicles consisted of oocytes surrounded by a single layer of rounded granulosa cells.
- Pre-antral follicles were defined as oocytes surrounded by more than 2 layers of granulosa cells, with either no antrum, or one to two small antral spaces.
- Antral follicles were characterised by the presence of a single large antral space.
- Preovulatory follicles typically have a single large antrum and the oocytes are surrounded by layers of cumulus cells attached to the end of a stalk of mural granulosa cells.
- the total number of primordial, primary, early antral, antral, and pre-ovulatory follicles was counted in every fifth ovarian section by light microscopy. To estimate the number of follicles at each classified stage in the whole ovary, the mean number of follicles per section was multiplied by a factor of 5, as only every fifth section was examined. A Student T test was used to determine the statistical significance of follicle populations between SPRASA and control KLH immunised females.
- SPRASA antibodies may cause profound infertility and that the presence of these antibodies even when fertilization occurs (i.e. at an antibody level below the level at which it causes infertility) may cause pregnancy pathologies such as fetal death, recurrent miscarriage, as well as causing uterine adhesions and abnormalities of the uterus.
- Detection of SPRASA antibodies in infertile individuals may be a useful indicator of the cause of their infertility and a useful indicator of appropriate treatment choices.
- Such treatments might include, but would not be limited to, intracytoplasmic sperm injection (ICSI), immunosuppression, in vitro fertilisation, intrauterine insemination.
- ICSI intracytoplasmic sperm injection
- immunosuppression in vitro fertilisation
- intrauterine insemination intrauterine insemination.
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Abstract
The present invention generally relates to methods and kits for identifying the cause of infertility and furthermore identifying the likelihood of an infertile subject responding to infertility treatment based on levels of antibodies reactive with Sperm Protein Reactive with Antisperm Antibodies (SPRASA, also known as SLLP- 1 and SPACA-3) in a sample from an infertile subject.
Description
METHODS AND KITS FOR INFERTILITY TESTING
FIELD OF THE INVENTION
The invention generally relates to methods and kits for identifying the cause of infertility and furthermore identifying the likelihood of an infertile subject responding to infertility treatment.
BACKGROUND OF THE INVENTION
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Not all couples who wish to achieve pregnancy will naturally be able to do so.
Some couples will require medical intervention to resolve underlying infertility problems. Infertility relates to the biological inability to conceive after 12 months of regular unprotected intercourse. The World Health Organisation has identified infertility as a public health issue and there is a need for more research in this area.
With respect to potential cause of infertility, antisperm antibodies (ASA) have been reported in infertile couples (Ayvalitois et al., 1985; Collins et ah, 1993), but also in fertile men and women (Heidenreich et al., 1994; Sinisi et al., 1993; Omu et al., 1997). This apparent contradiction indicates that not all ASA cause infertility. The uncertainty of the correlation between ASA and infertility is further compounded by the lack of standardised diagnostic tests, and their subsequent interpretation (Mazumdar & Levine, 1998). Some antisperm antibodies, although reactive in these diagnostic tests may be irrelevant to infertility. Current tests do not detect ASA directed against specific fertilisation-related antigens (Bronson, 1999), which may impair processes leading to successful fertilisation.
Thus, despite extensive clinical investigations the cause of infertility remains unexplained for many infertile couples and consequently they may not receive the ideal treatment. In vitro fertilisation (IVF) treatment is one approach taken by many couples that have difficulty in conceiving. This process can be long, arduous and extremely costly. Not all couples that choose this treatment will successfully fall pregnant. Prior to IVF treatment, patients are also required to undergo a number of routine
investigations and procedures, additive to the cost of IVF treatment.
Recent in vitro studies have shown that antibodies reactive with Sperm Protein Reactive with Antisperm Antibodies (SPRASA, also known as SLLP-1 and SPACA-3), a highly conserved sperm protein that has recently been isolated from infertile men
(Chiu, Erikson et al. 2004), inhibit sperm-oocyte binding in a zona- free hamster oocyte binding assay (Mandal, Klotz et al. 2003) which indicates that SPRASA plays an important role in fertilization. Further experimental work suggests that SPRASA is expressed by oocytes and maybe expressed by preimplantation embryos (Wagner, A., et al, 2006; Wagner, A. et al, 2008). There are at least two iso forms of the SPRASA protein, the long iso form of the protein is encoded by exons 1-5 of the gene, while a short iso form is encoded only by exons 2-5. The long iso form appears to be expressed only in sperm of higher primates while the short isoform is more widely expressed and is present in the sperm and oocytes of many mammalian species (Prendergast, Woad et al. 2008; Prendergast, Woad et al. 2008). Expression of SPRASA by both oocytes and sperm suggests that this protein has an important function in fertility. However, there is no information on the incidence of SPRASA antibodies in humans, and whether there is a correlation between SPRASA-reactive antibodies and infertility.
It would therefore be desirable to have an inexpensive and simple test in place that identifies the likelihood of a patient to respond to conventional infertility treatments, and thus help the patient (and health care provider) make an informed decision whether or not to proceed with costly infertility treatments.
It would also be desirable to have a test that would help determine the cause of infertility.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
SUMMAR Y OF THE INVENTION
The present invention relates broadly to a prognostic screen of women seeking fertility treatment to exclude those who have SPRASA reactive antibodies (and who would be expected to respond less well to some infertility procedures/treatment than others).
According to a first aspect, the present invention provides a method of identifying the likelihood of an infertile subject responding to infertility treatment comprising:
1) contacting a body fluid sample obtained from the subject with SPRASA
antigen or a functional part thereof and
2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated level of SPRASA reactive antibodies in said sample, when compared to control levels, indicates that the infertile subject is not likely to respond to infertility treatment.
According to a second aspect, the present invention provides a method of diagnosing cause of infertility in a subject comprising:
1) contacting a body fluid sample obtained from the subject with SPRASA antigen or a functional part thereof and
2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated level of SPRASA reactive antibodies in said sample, when compared to control levels, is indicative of the cause of infertility.
According to a third aspect, there is provided a method of determining the predisposition of a subject to developing a pregnancy pathology comprising
1 ) contacting a body fluid sample obtained from the subject with SPRASA antigen or a functional part thereof and
2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated levels of SPRASA reactive antibodies in said sample, when compared to control levels, indicates that the subject is predisposed to developing a pregnancy pathology.
Pregnancy pathologies can include but are not limited to foetal death, recurrent miscarriage, uterine adhesions, abnormalities of the uterus and the like.
Preferably the subject is a human and even more preferably the subject is a female human subject. Of course it will be understood that due to high level of conservation of the SPRASA protein across species the methods of the present invention are equally useful and applicable to other species.
Preferably the body fluid collected from the infertile subject is blood, which may be processed to plasma or serum, or may include follicular fluid, cervical mucus, semen or seminal plasma or uterine washings.
Preferably the SPRASA reactive antibody to be measured and/or detected is IgG but IgA or IgM antibodies may also be detected/measured using the same methods and assay formats. The antibody to be detected in the body fluid sample may either be a free antibody or it may be complexed to the SPRASA antigen. For convenience, the
"control" level of antibody may be set by measurements of antibody levels in blood of a population of fertile subjects. However, it will be understood that control levels of
antibody may be determined in any other suitable body fluid sample and population of subjects, depending on requirements.
Conventional infertility treatment can be selected from in vitro fertilisation (IVF), intracytoplasmic sperm injection (ICSI), intrauterine insemination, ovulation induction, hormone treatment and the like.
According to a fourth aspect, there is provided a kit for detecting and/or measuring anti-SPRASA antibodies in a fluid sample, comprising SPRASA antigen or a functional part thereof and reagents for detection of anti-SPRASA antibodies or anti- SPRASA antibody/SPRASA complex.
The test kits of the present invention may include a porous surface or solid substrate to which the antigen has been preabsorbed or covalently bound, said surface substrate being preferably in the form of microtitre plates or wells; test sera; various diluents and buffers; labelled conjugates for the detection of specifically bound antibodies and other signal-generating reagents such as enzyme substrates, co factors and chromogens.
Conveniently the SPRASA antigen or protein, or a functional part thereof, can be obtained by recombinant or synthetic means. Thus, recombinant SPRASA product can span individual or multiple exons of the SPRASA coding sequence. It will be understood that fragments, motifs and mimetics of SPRASA that have functional or specific antigenic activity can also be used.
The reagents for detection of anti-SPRASA antibody or anti-SPRASA
antibody/SPRASA complex may be selected from those conventionally used in the Enzyme-linked immunosorbent assay (ELISA) format, for example labelled (e.g.
biotinylated) heterologous species anti-IgG, IgA or IgM antibodies. In an RIA format assay such heterologous species antibodies can be labelled with a radionuclide (e.g. 131I, 3H or similar). In a fluorescent assay system detection antibodies may be labelled with a varity of fluorophores for example, fluorescene isothiocyanate (FITC). Other useful assay formats will be clear to those skilled in the art.
Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".
BRIEF DESCRIPTION OF THE DRA WINGS
Figure 1 : Photograph of purified recombinant SPRASA (exons 2-5, short isoform) band at 15kDa .
Figure 2: Photograph of purified recombinant SPRASA(exonsl -5, long isoform) band at 26 kDa.
Figure 3: IgG immunoreactivity of infertile and fertile women against recombinant SPRASA exons 2-5 (short isoform). Infertile patients M27, 45 and 48 have higher levels of SPRASA reactive IgG antibodies than other infertile and fertile women.
Figure 4: IgG immunoreactivity of sera from men and women in the fertile and infertile groups against recombinant SPRASA exons 1-5 (long isoform). Horizontal line indicates the cut-off level for high anti-SPRASA antibody levels. (0.8 is the value for the cut-off level as determined by normalising values of the test samples against the values obtained for corresponding reference samples included in each ELISA performed. The test samples are expressed as a percentage of the median of the reference samples.) - 13 infertile individuals and 1 fertile individual are shown to have anti-SPRASA antibody levels above the cut-off. Of the infertile individuals 6 were men and 7 were women. 4 of these individuals had otherwise unexplained infertility.
Figure 5: Mean uterine weights of females immunised with SPARS A and keyhole limpet peptide (KLH), respectively, and mated correspondingly with males immunised subcutaneously (s.c.) with SPRASA and KLH, respectively, at 12 days post- coitus (dpc). Data are expressed as mean + standard deviation (SD). There were no significant differences in the uterine weights between SPRASA and KLH immunised females.
Figure 6: Mean litter size of females immunised with SPARSA and KLH, respectively, and mated correspondingly with males immunised s.c. with SPRASA and KLH, respectively, at 12 dpc. Data are expressed as mean + SD. There were no significant differences in the litter size between SPRASA and KLH immunised females.
Figure 7: Mean individual pup weights of females immunised with SPARSA and KLH, respectively, and mated correspondingly with males immunised (s.c.) with SPRASA and KLH, respectively, at 12 dpc. Data are expressed as mean + SD. There were no significant differences in the individual pup weight between SPRASA and KLH immunised females.
Figure 8: Representative photographs of uteri from (A) SPRASA and (B) KLH immunised females at 12 dpc. Uteri from SPRASA immunised females appeared
distinctly different from the uteri from KLH immunised females. Embryo clustering and reabsorptions seen in SPRASA immunised females are indicated by black and white arrows, respectively.
Figure 9: Follicle count per ovary in SPRASA and KLH immunised females, respectively. Data are expressed as mean + SD. There were significant fewer primordial follicles in SPRASA immunised females compared to KLH immunised females (*p=0.0028). There were no significant differences in the number of follicles at any other development stage between SPRASA and KLH immunised females. Dotted bars = KLH immunised females and lined bars = SPRASA immunised females.
Figure 10: Mean litter size of 10 Wistar rats immunised against recombinant
SPRASA (short isoform; exons 2-5) were significantly ( p 0.0268) smaller than the mean litters size of control Wistar rats immunised against KLH. Both groups of immunised female were mated with unimmunised proven stud males.
Figure 1 1 : Shows the mRNA nucleotide sequence of exons 1 to 5 of the mouse (A; gi| 1 10626053|ref]NM_029367.11 Mus musculus sperm acrosome associated 3 (Spaca3), mRNA) and the rat SPRASA gene (B; gi| 157786663|ref|NM_001 105820.1 | Rattus norvegicus sperm acrosome associated 3 (Spaca3), mRNA). Exons are indicated by alternating underlined and bold nucleotides (i.e. in Figure 1 A, the sequence of exon 1 is underlined, the sequence of exon 2 is in bold text, the sequence of exon 3 is underlined again, and so forth); the start ATG codon is indicated by a white box.
Figure 12: Shows the mRNA nucleotide sequence of exons 1 to 5 of the human SPRASA gene (gi| 142360775|ref|NM_173847.3| Homo sapiens sperm acrosome associated 3 (SPACA3), mRNA) (Exons and start ATG codon are indicated as in Figure 11 above).
Figure 13: Shows an alignment of the human, mouse and rat SPRASA exon sequences. The start ATG codon is indicated as bold and underlined nucleotides.
PREFERRED EMBODIMENT OF THE INVENTION
The present invention is based in part on the studies which demonstrate that not only is SPRASA expressed by oocytes, where it is present on the oolemmal membrane and in the zona pellucida, but that some infertile subjects have a circulating antibody reactive with SPRASA.
Antisperm (and anti-oocyte) antibodies have long been known to be associated with infertility but these antibodies may also be found in fertile persons. This limits somewhat the diagnostic/prognostic value of antisperm antibody testing. This is in part
because current tests do not distinguish between sperm/ovarian antigens that are crucial for fertility and those that are not.
The present results show SPRASA is crucial for fertility in both humans, mice and rats. 3% of infertile women had elevated levels of antibodies reactive with the short SPRASA isoform (exons 2-5; when compared to corresponding antibody levels in a population of fertile women), while 13 individuals (6 men and 7 women) had antibodies reactive with the long SPRASA isoform (exons 1-5; when compared to corresponding antibody levels in a population of fertile individuals). Of these 13 individuals four were from couples with otherwise unexplained infertility. Female mice immunised with SPRASA (exons 2-5; short isoform) were profoundly infertile while female rats immunised with SPRASA (exons 2-5; short isoform) had significantly smaller mean litter sizes than control immunised female rats (Figure 10). This suggests that, in affected couples, SPRASA antibodies may be a dominant cause of their infertility.
The methods and kits of the present invention, preferably based on the ELISA format, are simple and inexpensive diagnostic and prognostic tools, effective in identifying causes of infertility in women, informing treatment decisions or at least informing possible lack of response to conventional infertility treatments. An added advantage of the methods and kits of the present invention is that certain pregnancy complications and pathologies (including miscarriage and still birth) can be predicted should fertilisation take place, based on measurement of circulating anti-SPRASA antibody or anti-SPRASA antibody/SPRASA complex.
Although the percentage of men and women who would be excluded or directed away from conventional infertility treatments would be relatively small, the cost saving in doing so would still outweigh the cost of screening all women seeking infertility treatment. Women who are directed away from conventional treatments may instead be directed to a particular type of treatment, for example, directly to intracytoplasmic sperm injection (ICSl) rather than having several IVF cycles before having ICSL Alternatively they may be directed away from intrauterine insemination (IUI) and directed to ICSl. Immunosuppression which has been largely abandoned as a conventional fertility treatment may be appropriate for infertile subjects affected by SPRASA antibodies.
Preferred embodiments of the invention will now be described, with reference to non-limiting examples and figures.
EXAMPLES
1
The techniques and procedures are generally performed according to
conventional methods in the art and various general references (see, generally,
Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y, USA). Reagents have been sourced from Sigma Chemical Co. (USA), Life technologies (USA) or BioRad
Laboratories (USA) unless indicated otherwise.
EXAMPLE 1 - Expression of Histidine-tagged Recombinant Human SPRASA (short isoform; exons 2-5)
PCR products (Forward primer: aaagtggatccatgttgttggccctggtctgt and Reverse primer: cccgggaattctca gaagtcacagccatccaccca) were cloned into a protein expression vector (pPROX HTb), and the recombinant plasmids were transformed in E.coli, and sequenced to confirm the cloned products. Single colonies of E.coli expressing the recombinant plasmids were grown at 37°C in an overnight culture of 10ml Luria broth (LB) containing 100μg/ml ampicillin, and used to inoculate 2L of pre-warmed LB media.
Recombinant SPRASA expression was induced with the addition of ImM Isopropyl-P-D-thiogalactopyranoside (IPTG) and the culture was incubated overnight at 37°C. Cells were harvested by centrifugation at 8,000 x g for 20 minutes, and the resultant pellet resuspended in Tris base buffer containing 8M urea (Buffer A), pH = 8. Cells were lysed by a cell disruptor at 18 ksi, and the soluble and insoluble fractions separated by centrifugation at 15,000 x g for 20 minutes. The supernatant was discarded, and the insoluble pellet was dissolved in Buffer A with shaking on a rotator. Supernatant from the insoluble fraction was then filtered and loaded onto a 5ml Ni- charged Hi-Trap chelating column. The column was washed with 20ml of Buffer B
(TBS buffer containing 8M urea and 50mM Imidazole, pH = 8). Recombinant SPRASA was then eluted with 100ml of elution buffer with an increasing gradient of Imidazole (50mM to 500mM), at a flow rate of lml/min, collected in 2ml fractions.
EXAMPLE 2A- Purification of Recombinant Human SPRASA (short isoform; exons 2-5)
The purity of eluted Histidine-tagged recombinant SPRASA was evaluated by Coomassie Blue staining following SDS PAGE, and by western blotting. 20 ί from each of the 2ml fractions were mixed with equal volume of 2x sample buffer and separated on a 15% polyacrylamide gel in a Mini-Protean II Cell at 120V under reducing
conditions (10% 2-mercaptoethanol). Protein bands were then stained with Coomassie Brilliant Blue G250 to detect a band at the predicted molecular weight (approximately 15 kDa) or transferred to a 0.45μπι nitrocellulose membrane (Hybond-C extra,
Amersham, Auckland, NZ) using a semi-dry transfer apparatus for 35 minutes at 2mA for western blotting. Following the transfer, non-specific binding sites on the membranes were blocked with 5% non-fat milk in PBS-Tween for an hour at room temperature, followed by incubation with polyclonal SPRASA antibody at 1 : 100 dilution in blocking solution for an hour. After three 10 minute PBS-Tween washes, the membranes were incubated with biotinylated anti-rabbit IgG (Jackson Laboratories, USA) at 1 :2000 dilution in blocking solution for an hour. Membranes were then washed three times in PBS-Tween and incubated for an hour with horseradish peroxidase (HRP) conjugated streptavidin (Zymed Laboratories, USA) at 1 :5000 dilution in blocking solution. After three further PBS-Tween washes, the signals were detected by enhanced chemiluminescence (Amersham, Auckland), with 1 minute X-ray film exposure time, followed by processing in an automated film developer (Agfa Curix60, Germany).
Since these analyses showed that recombinant SPRASA was not pure, the recombinant protein was further purified by elution of the protein following
electrophoretic separation of fractions containing SPRASA. To purify recombinant SPRASA, Ni-affinity chromatography fractions containing SPRASA were pooled and separated on a 15% polyacrylamide gel as described previously. Two thin strips were excised from either side of the gel and stained with Coomassie Blue to identify protein bands at the predicted molecular weight of recombinant SPRASA (15kDa). Following staining, the strips were destained in destaining solution, and rehydrated in tap water. The strips were then used as a guide for excising the protein band at 15kDa on the unstained gel. The excised gel strip was minced and left overnight at 4°C in 8M urea in cold PBS. The following morning, the supernatant was aspirated and concentrated using a lOkDa centrifugal concentrator (Vivaspin, Sartorius, Auckland, NZ), at 5000 x g. Purified and concentrated recombinant SPRASA was stored at -20°C until required. Aliquots containing 20μg of purified recombinant SPRASA were re-run on a 15% polyacrylamide gel, as described previously, to confirm the purity of the concentrated sample. Result was a single band at 15kDa (Figure 1).
EXAMPLE 2B - Expression and purification of Histidine-tagged Recombinant Human SPRASA (long isoform; exons 1-5)
A recombinant SPRASA gene (exons 1-5) lacking the trans-membrane region (amino acids MLLALVCLLSCLLPSSEA of wild-type SPRASA) and having an N- terminal hexa-histidine sequence (optimised for codon usage in E. coli) was produced by GenScript of Wheelock House, Level 18, 20 Pedder Street, Central, Hong Kong. The construct was cloned into the plasmid pUC57. The DNA sequence of the SPRASA construct was:
ATGGTTTCTGCTCTGCGTGGTGCCCCGCTGATTCGTGTTCAT TCATCTCCGGTCTCCTCTCCGAGTGTTAGTGGTCCGCGCCGC CTGGTTAGCTGCCTGAGCTCTCAGAGTTCCGCCCTGTCACAA TCGGGCGGTGGCAGCACCTCTGCAGCAGGTATTGAAGCACG TTCTCGTGCACTGCGTCGCCGTTGGTGCCCGGCAGGTATCA AACTGTATGGTCGCTGTGAACTGGCACGTGTCCTGCATGAT TTTGGTCTGGACGGCTATCGCGGTTACTCACTGGCTGATTGG GTGTGTCTGGCGTATTTTACCTCGGGTTTCAACGCAGCTGCG CTGGATTACGAAGCTGACGGCTCAACGAACAATGGTATTTT CCAGATCAACAGTCGCCGTTGGTGCTCCAATCTGACGCCGA ACGTGCCGAATGTTTGCCGTATGTATTGTTCCGATCTGCTGA ACCCGAATCTGAAAGACACCGTCATTTGTGCGATGAAAATC ACGCAGGAACCGCAAGGTCTGGGCTACTGGGAAGCGTGGC GTCATCACTGTCAAGGCAAAGACCTGACCGAATGGGTGGAT GGCTGCGATTTC
This expression construct encodes for a protein having the following amino acid sequence:
Initiating Methionine of SPRASA
MHHHHHHENLYFQG MVSALRGAPLIRVHSSPVSSPSVSGPRR
LVSCLSSQSSALSQSGGGSTSAAGIEARSRALRRRWCPAGI*KL
YGRCELARVLHDFGLDGYRGYSLADVCLAYFTSGFNAAALDY
EADGSTN GIFQINSRRWCSNLTPNVPNVCRMYCSDLLNPNLK
DTVICAMKITQEPQGLGYWEAWRHHCQGKDLTEWVDGCDF
"*" In the above amino acid sequence indicates where amino acids of the transmembrane domain would appear in wild-type SRASA.
This protein was expressed in E. coli and purified from inclusion bodies by nickel chlorine affinity chromatography as described for histidine-tagged recombinant short SPRASA isoform (exons 2-5; see Example 2A above). After solubilisation of the
inclusion bodies the protein was resuspended in phosphate buffered saline containing 0.2% sodium lauroyl sarcosine, pH 8.0.
EXAMPLE 3 - Screening for SPRASA-Reactive Antibodies in Infertile Patients Collection of Blood Samples
Fertile and infertile patients recruited for this investigation were accessed through the maternity services of National Women's Hospital following radio advertisements, and Fertility Plus (the fertility service of Auckland District Health Board, NZ), respectively, as well as through Fertility Associates. Blood samples were allowed to clot overnight at 4°C and processed the following morning by centrifugation, at 2400 x g for 10 minutes. Serum was aspirated, aliquoted and stored at -80°C until required.
A) ELISA Screening for short SPRASA isoform (exons 2-5) -Reactive Antibodies in Patient Serum
In order to determine whether SPRASA-reactive antisperm antibodies are associated with human infertility, purified recombinant human SPRASA (short isoform; exons 2-5) was used as the antigen in an ELISA to screen the serum of 102 infertile couples, as well as 104 fertile control couples. High affinity binding ELISA plates (Corning, Auckland, New Zealand) were incubated overnight at 4°C with 40μΙ, of recombinant SPRASA at a concentration of 1 μg/ml in each well, diluted in carbonate buffer (0.1M, pH 9). The plates were washed three times with PBS-Tween to remove unbound recombinant SPRASA. Non-specific binding sites were blocked with 40μ1 of 5% non-fat milk powder in PBS-Tween for an hour at room temperature. After three washes with PBS-Tween, the plates were incubated with patient sera at 1 : 100 dilution in blocking solution for an hour at room temperature. The plates were then washed three times in PBS-Tween, and incubated with biotinylated mouse anti-human IgG or IgA, diluted 1 :2000 in blocking solution for an hour at room temperature. After three washes in PBS-Tween, the plates were incubated with Horseradish peroxidase (HRP)- conjugated streptavidin at 1 :5000 dilution in blocking solution for an hour, followed by three more washes in PBS-Tween. To measure enzyme activity, 40μ1 of TMB substrate was added to each well, and the colour reaction was allowed to develop at room temperature in the dark for 30 minutes. The reaction was stopped by adding 20μ1 of
2molL"' H2S04, and absorbance at 450nm was quantified using a Benchmark microplate reader. Serum samples were run in duplicate, and on each plate, 7 randomly selected fertile controls were run to allow normalization. The median OD value from the 7 fertile controls was used to normalize the OD values in each ELISA plate, and each individual test sample was expressed as a value derived from the OD value of the individual over the median OD value from the controls.
Statistical Analysis of SPRASA- Reactive Antibodies in Infertile and Fertile Serum Samples
Normalized OD values of infertile and control fertile patients were plotted using box plots. The box plots show the distribution of the data with the mean, median and outliers. The SPRASA-reactive IgG data was positively skewed. The data was therefore logarithmically transformed to reduce any association between the mean and the variance. A Student's T test was used to assess the significance of SPRASA-reactive IgG antibodies in female fertility.
Results - Detection of SPRASA Antibodies in the Sera of Infertile and Fertile Patients
In order to determine whether SPRASA-reactive antibodies were correlated with infertility, an ELISA was used employing recombinant SPRASA as an antigen. Infertile and fertile control couples were screened for SPRASA-reactive IgG antibodies. This analysis showed that women, regardless of fertility status had significantly higher levels of SPRASA IgG levels than men in the infertile or fertile control groups (p<0.001) (Figure 2). The level of SPRASA IgG was elevated in infertile women compared to fertile women, however, this difference was not significant (p=0.06). However, there were three women (M27, M45, and M48) in the infertile group that had more elevated IgG values of >2.2 (Figures 2 and 3). There was no correlation between SPRASA IgG levels and fertility status in men (p>0.05).
B) ELISA Screening for long SPRASA isoform (exons 1-5) -Reactive Antibodies in Patient Serum
For the detection of antibodies against the long SPRASA isoform (exons 1-5), high affinity binding ELISA plates were incubated overnight at 4°C with 50μ1 of recombinant long SPRASA isoform (exons 1-5) at a concentration of 20μg/ml well diluted in carbonate buffer (0.1M, pH9). In order to remove unbound SPRASA, plates were washed three times using PBS-Tween. To prevent non-specific binding plates were blocked with 50μ1 of 5% non-fat milk powder in PBS-Tween at room temperature. After
three washes, the plates were incubated with patient sera (1 : 100 dilution in blocking solution) for an hour at room temperature. Following three washes in PBS-Tween, plates were incubated with biotinylated mouse anti-humans IgG or IgA (1 :2000 dilution in blocking solution) for an hour at room temperature.
After three more washes in PBS-Tween. the plates were incubated with horseradish peroxidase (HRP)-conjugated streptavidin at 1 :2000 (for IgG assay) orl :5000 (for IgA assay) in blocking solution for an hour, followed by three more washes in PBS-Tween. To measure enzyme activity, 50μ1 of 0.1% o-phenylenediamine dihydrochloride (OPD) substrate solution was added to each well. The reaction was stopped using 10% HC1 and absorbance at 490nm wavelength was measured and recorded using a Benchmark microplate reader. Screening was performed in duplicate. Samples were excluded if coefficient of variation was above 10%. Results were normalised to the median of nine negative control samples which were included in each plate.
Results - Detection of long SPRASA isoform (exons 1-5) - Antibodies in the Sera of Infertile and Fertile Patients
A total of 189 infertile individuals (99 males and 90 females), as well as 196 fertile individuals (104 males and 92 females) were included in the analysis for IgG anti- SPRASA antibodies. An arbitrary cut-off value (Normalised Value of 0.8) was assigned to the results of the IgG assay which identified 14 individuals as having high levels of reactive long SPRASA-isoform (exon 1 -5) antibodies (1 fertile and 13 infertile individuals). Of the 13 infertile individuals, high levels of reactive long SPRASA- isoform (exon 1 -5) antibodies were found in 6 individuals who were partners in 3 respective couples, and in 3 additional males and 4 additional females.
EXAMPLE 4 - Immunisation of female mice with recombinant human SPRASA (short isoform; exons 2-5)
To investigate the effect of SPRASA antibodies on fertility, CD1 , 4 week old male (n=12) and female (n=12) mice weighing approximately 20g were immunised subcutaneously (s.c.) with purified recombinant SPRASA protein at two sites. Control mice were immunised with keyhole limpet peptide (KLH). Each animal received at least three immunisations at two week intervals, and each immunisation consisted of 50uL recombinant SPRASA protein (25μg) or KLH peptide (25μg) in PBS, emulsified with 50μΙ, of adjuvant. The first immunisation was given with Freund's Complete adjuvant, and subsequent booster immunisations employed Freund's Incomplete adjuvant.
A group of subcutaneously immunised males (n=6) also received uni-lateral intra- testicular immunisations with recombinant SPRASA. Control males (n=6) received the KLH carrier protein. Each animal was immunised four times at two weekly intervals, with ΙΟμΙ. aluminium hydroxide suspension containing 5μg of recombinant SPRASA or KLH.
Two weeks after the final immunisation, blood was obtained from the tail vein and tested for the level of SPRASA antibodies using the ELISA protocol.
Mating Trials
Trial 1) To determine the effect of SPRASA antibodies in females, immunised females (n=6) were date-mated with untreated males with proven fertility (n=3), commencing 2 months after the final immunization.
Trial 2) To further examine the effect of SPRASA antibodies in females, immunised females (n=6) were date-mated with males (n=3), commencing 6 months after the final immunization.
For both trials the methodology was then as follows; after placing the females and males together, females were examined the following morning for the presence of vaginal plugs to confirm coitus. Since not all females would present with vaginal plugs after coitus, all females regardless of the presence of vaginal plugs were housed separately from males the morning after. The weight of females was recorded every morning until day 12 to confirm pregnancy. Pregnant control females at 12 dpc typically gained 8 grams. At 12 dpc, pregnant females were euthanised by rising C02. Uterine weight, litter size, and individual pup weight were then measured. Females that did not become pregnant, were selected for mating at a later date when they came into oestrus, for a maximum of five times. A Student T test was used to determine the statistical significance of male and female fertility outcomes between SPRASA and KLH immunisations.
Results - The Effect of immunising mice with SPRASA on Female Fertility
To evaluate the effect of SPRASA antibodies in females, female CD1 mice were subcutaneously immunised with SPRASA (n=5), or control females were immunized with KLH (n=5) then date-mated with males with proven fertility (n=3) two months after the final immunisation. Although the presence of vaginal plugs provides an indication that mating has occurred, not all mated females present with vaginal plugs. All mated females, therefore, regardless of whether a vaginal plug was found, were monitored daily until 12 dpc. At day 12 dpc, if successfully mated, females gained approximately 8
grams, they were palpitated to confirm pregnancy. Pregnant females were then euthanised, and the uterine weight, litter size and individual pup weight were recorded as measures of fertility. Any females that did not become pregnant were mated again until a total of five matings had been completed.
The fertility of SPRASA immunised females was completely inhibited as immunised females were unable to become pregnant despite mating five times with males of proven fertility (Table 1). KLH immunised females in contrast became pregnant after an average of two matings with males of proven fertility (Table 1).
Table 1 showing the total number of matings to achieve successful pregnancy in
SPRASA and control (KLH) immunised females.
The effect of delayed mating in Female Mice immunised with SPRASA
To evaluate the effect of delayed mating in SPRASA or control immusined females, female CD1 mice were subcutaneously immunised with SPRASA (n=5), or control females were immunized with KLH (n=3) then date-mated with males (n=3) six months after the final immunisation. All mated females regardless of vaginal plug formation were monitored until 12 dpc for an increase in body weight of approximately 8 grams. Pregnant females, confirmed by palpitation were then euthanised for fertility analysis. Mated females that were not pregnant were mated again up to four times.
Two out of five SPRASA immunised females became pregnant after the first mating, one after the second mating, and the remaining two females were not pregnant after four matings. One out of three control KLH immunised females became pregnant after the first mating, and the remaining two females became pregnant after the second mating. On day twelve of pregnancy, there was a 32.7% reduction in the mean uterine weight of SPRASA immunised females compared to KLH immunised females, however this difference was not significant (p=0.136) (Figure 4). The mean number of pups was similar between SPRASA and KLH immunised females (p=0.685) (Figure 5). There was a 25.6% reduction in the mean individual pup weight of SPRASA immunised females compared to the mean pup weight of KLH immunised females, however, this difference was not significant (p=0.285) (Figure 6).
It is worth noting that removal of uteri from pregnant SPRASA immunised females was difficult, as all uteri (n=3) were adherent tightly to neighbouring structures
within the abdominal cavity. On observation, uteri from SPRASA immunised females appeared to be encased by thick layers of serosa/connective tissue. In addition, pups within the uteri were not distinct structures and a number of embryo reabsorptions were observed (Figure 7). In contrast, removal of uteri from KLH immunised females was performed without difficulty, as uteri were not adherent to neighbouring structures in the abdominal cavity. Uteri from KLH immunised females also appeared normal, without the thickening of connective tissue observed in SPRASA immunised females. Finally, embryo reabsorptions were not observed in pregnant KLH immunised females (Figure 7). EXAMPLE 5 - Histological Examination of Ovaries from SPRASA (short isoform; exons 2-5) Immunised Female Mice
Ovarian tissue from SPRASA and control immunised female mice in dioestrus were excised and fixed in 4% PFA overnight at 4°C. The following morning, ovaries were dehydrated in 70% ethanol at room temperature, cleared in xylene (BDH,
Auckland), and embedded in paraffin. Serial ovarian sections (6um) were cut using a microtome onto poly-l-lysine coated slides, and allowed to air-dry. Slides were incubated at 55°C for 10 minutes, or overnight to melt the paraffin, followed by two changes of xylene, 10 minutes each. Prior to staining with haematoxylin, slides were submersed in a series of rehydrating baths of 100%, 95%, 70%, 50% and 30% ethanol diluted in water (v/v) for 2 minutes each, and left in deionised water for 4 minutes.
Follicle populations in the ovaries of SPRASA and control KLH immunised females were evaluated using light microscopy. The follicle classification system employed was described previously by Myer et al., 2004:
• primordial follicles are characterised by oocytes surrounded by a flattened
granulosa cell layer.
• Primary follicles consisted of oocytes surrounded by a single layer of rounded granulosa cells.
• Pre-antral follicles were defined as oocytes surrounded by more than 2 layers of granulosa cells, with either no antrum, or one to two small antral spaces.
• Antral follicles were characterised by the presence of a single large antral space.
• Preovulatory follicles typically have a single large antrum and the oocytes are surrounded by layers of cumulus cells attached to the end of a stalk of mural granulosa cells.
The total number of primordial, primary, early antral, antral, and pre-ovulatory follicles was counted in every fifth ovarian section by light microscopy. To estimate the
number of follicles at each classified stage in the whole ovary, the mean number of follicles per section was multiplied by a factor of 5, as only every fifth section was examined. A Student T test was used to determine the statistical significance of follicle populations between SPRASA and control KLH immunised females.
Results - Quantification of Ovarian Follicles in SPRASA Immunised Female Mice There is evidence that active immunisation with zona pellucida proteins is associated with anti-fertility effects, mediated by 2 mechanisms: (1) that serum antibodies directed against the immunised protein inhibit sperm-oocyte interactions by blocking receptor sites, and (2) that serum antibodies interfere with normal ovarian function by disrupting folliculogenesis. One of the metabolic consequences of loss of ovarian function in ovariectomised mice is increased adipose tissue deposition (Stoops et al., 2006). In order to examine ovarian function, the ovaries of three SPRASA and three KLH immunised females were fixed in paraformaldehyde, paraffin embedded, and sectioned to completion. Every fifth section was examined for the presence of follicles at each development stage, and the number of follicles per ovary was calculated.
Histological examination of the ovaries revealed there was a significant reduction in the number of primordial follicles (*p=0.0028) in SPRASA immunised females compared to KLH immunised females. However, there were no significant differences in the number of follicles at other stages of development in SPRASA immunised females compared to KLH immunised females, including primary (p=0.987), early antral (p=0.285), late antral (p=0.566), and pre-ovulatory (p=0.446) follicles (Figure 8).
Corpora leutea were also consistently observed in both SPRASA and KLH immunised females.
EXAMPLE 6 - Immunisation of female rats with recombinant human SPRASA (short isoform; exons 2-5)
To further investigate the effect of SPRASA (short isoform; exons 2-5)
antibodies on fertility, female Wistar rats (n=10; approximately 6 weeks old; weighing approximately! 60-220g) were immunised subcutaneously with purified the recombinant short SPRASA isoform at two sites. Control rats were immunised with keyhole limpet peptide (KLH). Each animal received at least three immunisations at two week intervals, and each immunisation consisted of 200 μΐ recombinant SPRASA protein (10(^g) or KLH peptide (10(^g) in PBS, emulsified with 200μ1 of adjuvant. The first immunisation was given with Freund's Complete adjuvant, and subsequent booster immunisations employed Freund's Incomplete adjuvant.
Two weeks after the final immunisation, blood was obtained from the tail vein of each animal and tested for the level of SPRASA antibodies using the ELISA protocol described above (see example 3A).
Mating Trial
To determine the effect of SPRASA antibodies in females, immunised females
(n=10) were date-mated with untreated males with proven fertility (n=6), commencing 2-3 months after the final immunization. After placing the females and males together over night, females were examined the following morning for the presence of vaginal plugs to confirm coitus. If there was a plug the males and females were separated and at 12 dpc the females were palpated to confirm pregnancy. This was confirmed at 20 dpc and pregnant females were euthanaiased and dissected to allow determination of the litter size. If there was no plug the males and females were separated and the females were placed with a different study male when they next entered oestrous. A Mann- Whitney U test was used to determine the statistical significance of litter size difference outcomes between SPRASA and KLH immunised animals.
Results - The Effect of immunising rats with SPRASA on Female Fertility
All control females became pregnant on the first mating. Of the SPRASA- immunised females seven became pregnant on the first mating, two became pregnant after two matings and one required three matings to become pregnant. The mean litter size of the SPRASA immunised females was 12.1 pups which was significantly smaller than that of the control, KLH-immunised, females (16.4, p= 0.027; Figure 10).
Conclusions
The combination of data from our human and rodent studies suggest that elevated levels of antibodies reactive with SPRASA cause infertility that may not be detectable by examination of follicle counts except possibly at the level of primordial follicles.
The data suggest that elevated levels of SPRASA antibodies may cause profound infertility and that the presence of these antibodies even when fertilization occurs (i.e. at an antibody level below the level at which it causes infertility) may cause pregnancy pathologies such as fetal death, recurrent miscarriage, as well as causing uterine adhesions and abnormalities of the uterus.
Detection of SPRASA antibodies in infertile individuals may be a useful indicator of the cause of their infertility and a useful indicator of appropriate treatment choices. Such treatments might include, but would not be limited to, intracytoplasmic
sperm injection (ICSI), immunosuppression, in vitro fertilisation, intrauterine insemination.
Although the invention has been described with reference to specific example, will be appreciated by those skilled in the art that the invention may be embodied in many other forms.
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VI, J.L. Juengel, J.F. Murray, and M.F. Smith, Editors. 2006, Nottingham University Press: Wellington, New Zealand, p. 556.
Claims
1. A method of identifying the likelihood of an infertile subject responding to infertility treatment comprising:
1) contacting a body fluid sample obtained from the subject with Sperm Protein
Reactive with Antisperm Antibodies (SPRASA) antigen or a functional part thereof and 2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated level of SPRASA reactive antibody in said sample, when compared to control levels, indicates that the infertile subject is not likely to respond to infertility treatment.
2. The method of claim 1, wherein the infertility treatment is selected from in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), intrauterine insemination, ovulation induction and hormone treatment.
3. A method of diagnosing the cause of infertility in a subject comprising:
1 ) contacting a body fluid sample obtained from the subject with SPRASA antigen or a functional part thereof and
2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated level of SPRASA reactive antibody in said sample, when compared to control levels, is indicative of the cause of infertility.
4. A method of determining the predisposition of a subject to developing a pregnancy pathology comprising
1 ) contacting a body fluid sample obtained from the subject with SPRASA antigen or a functional part thereof and
2) detecting SPRASA reactive antibody levels in said sample,
wherein elevated levels of SPRASA reactive antibody in said sample, when compared to control levels, indicates that the subject is predisposed to developing a pregnancy pathology.
5. The method according to claim 4, wherein the pregnancy pathology is selected from foetal death, recurrent miscarriage, uterine adhesions, and abnormalities of the uterus.
6. The method according to any one of claims 1 to 5, wherein the SPRASA antigen is selected from recombinant SPRASA, or fragments, motifs and mimetics of SPRASA that have functional or SPRASA-specific antigenic activity.
7. The method according to any one of claims 1 to 6, wherein the SPRASA antigen is the long SPRASA isoform encoded by exons 1 to 5 of the SPRASA gene.
8. The method according to any one of claims 1 to 6, wherein the SPRASA antigen is the short SPRASA isoform encoded by exons 2 to 5 of the SPRASA gene.
9. The method of any one of claims 1 to 8, wherein the SPRASA reactive antibody to be detected is an IgG, IgA or IgM antibody.
10. The method of any one of claims 1 to 9, wherein the SPRASA reactive antibody to be detected is in a complex with the SPRASA antigen.
1 1. The method of any one of claims 1 to 10, wherein the body fluid sample is selected from blood, plasma, serum, follicular fluid, cervical mucus, semen or seminal plasma or uterine washings.
12. The method according to any one of claims 1 to 11, wherein the subject is a human.
13. The method according to claim 12, wherein the subject is a female.
14. A kit for detecting and/or measuring anti-SPRASA antibody or anti-SPRASA antibody/SPRASA complex in a fluid sample, comprising SPRASA antigen or functional part thereof and reagents for detection of anti-SPRASA antibody or anti- SPRASA antibody/SPRASA complex.
15. The kit of claim 14, wherein the SPRASA antigen is selected from recombinant SPRASA, or fragments, motifs and mimetics of SPRASA that have functional or antigenic activity.
16. The kit of claim 14 or claim 15, wherein the SPRASA antigen is the long
SPRASA isoform encoded by exons 1 to 5 of the SPRASA gene.
17. The kit of claim 14 or claim 15, wherein the SPRASA antigen is the short SPRASA isoform encoded by exons 2 to 5 of the SPRASA gene.
18. The kit of any one of claims 14 to 17, wherein the fluid sample is selected from blood, plasma, serum, follicular fluid, cervical mucus, semen or seminal plasma or uterine washings.
19. The kit of any one of claims 14 to 18, wherein the anti-SPRASA antibody to be detected and/or measured is selected from IgG, IgA and IgM antibodies.
20. The kit of any one of claims 14 to 19, wherein the reagents for detection of anti- SPRASA antibody or anti-SPRASA antibody/SPRASA complex are selected from those used in an ELISA format, RIA assay format or fluorescent assay system.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104977400A (en) * | 2015-07-27 | 2015-10-14 | 三明市和众生物技术有限公司 | Infertility joint detection kit and detection method thereof |
CN114133442A (en) * | 2021-12-02 | 2022-03-04 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPACA3 specific polypeptide and antibody |
Citations (1)
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WO2004064740A2 (en) * | 2003-01-16 | 2004-08-05 | University Of Virginia Patent Foundation | Sperm specific lysozyme-like proteins |
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2011
- 2011-06-28 WO PCT/NZ2011/000122 patent/WO2012005606A1/en active Application Filing
Patent Citations (1)
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WO2004064740A2 (en) * | 2003-01-16 | 2004-08-05 | University Of Virginia Patent Foundation | Sperm specific lysozyme-like proteins |
Non-Patent Citations (2)
Title |
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CHIU, W.W.C. ET AL.: "SPRASA, a novel sperm protein involved in immune-mediated infertility", HUMAN REPRODUCTION, vol. 19, 2004, pages 243 - 249 * |
CHIU, W.W.C. ET AL.: "Use of antisperm antibodies in differential display Western blotting to identify sperm proteins important in fertility", HUMAN REPRODUCTION, vol. 17, 2002, pages 984 - 989 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104977400A (en) * | 2015-07-27 | 2015-10-14 | 三明市和众生物技术有限公司 | Infertility joint detection kit and detection method thereof |
CN114133442A (en) * | 2021-12-02 | 2022-03-04 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPACA3 specific polypeptide and antibody |
CN114133442B (en) * | 2021-12-02 | 2024-02-02 | 天津市泌尿外科研究所 | Preparation method of human sperm membrane protein SPACA3 specific polypeptide and antibody |
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