Methods and polynucleotides and uses thereof

Abstract

This invention provides a method for stratifying subjects for treatment with human menopausal gonadotropin (hMG) or recombinant follicle-stimulating hormone (rFSH), the method comprising or consisting of the following steps: (a) providing a sample from the subject to be tested; (b) determining the follicle-stimulating hormone receptor (FSHR) N680S single nucleotide polymorphism (SNP) genotype of the subject; and (c) if the genotype is homozygous for the 680S SNP, stratifying the subject for hMG treatment; if the genotype is homozygous for N680 wild-type (WT), stratifying the subject for rFSH treatment. Methods and uses for treating involuntary infertility, infertility, and for reducing the risk of ovarian hyperstimulation syndrome; and related polynucleotide primers and kits for the above methods and uses.

Description

Technical Field

[0001] This invention relates to methods for stratifying involuntary childless individuals for appropriate treatment, as well as polynucleotides and kits useful in such methods. Background Technology

[0002] Assisted reproduction is widely used to treat infertility and involuntary childlessness. It is estimated that more than 2.5 million hormonal cycles are completed globally each year, and approximately 500,000 babies are born annually after assisted reproduction. To date, approximately 10 million children have been born through assisted reproduction, driven by childbearing delays and increased access to effective assisted reproductive technologies, particularly in developing countries. This number is expected to continue to grow in the coming years (World Health Organization, 2018, International Classification of Diseases, 11th revision; Mascarenhas). people, 2012, PLoS Med: 9(12); Boivin people, 2007, Hum Reprod: 22(6):1506-12).

[0003] The most commonly used fertilization technique is intracytoplasmic sperm injection (ICSI), which involves injecting a single sperm into eggs retrieved from a female partner, followed by selection of the best embryos for transfer. Overall, ICSI accounts for approximately 65% ​​of all treatments worldwide, while conventional in vitro fertilization (IVF), in which oocytes are retrieved through natural fertilization, makes up the remaining 35%. These percentages vary significantly between countries, but regardless of the technique used, female partners initially undergo what is known as controlled ovarian hyperstimulation, achieved through the gonadotropin follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) agonist human chorionic gonadotropin (hCG), which releases multiple follicles and subsequently matures multiple oocytes.

[0004] The optimal number of mature follicles ready to release oocytes is 10-15, but the range can be from zero to over 20. A number exceeding 20 is defined as ovarian hyperstimulation syndrome (OHSS), the most concerning iatrogenic side effect of this therapy. In addition to polyfollicular ovaries, severe OHSS is characterized by enlarged ovarian volume, abdominal pain, increased vascular permeability, and leakage of intracellular fluid into the extracellular space, leading to hemoconcentration and an increased risk of thrombosis. These women require medical intervention including parenteral fluid administration, drainage of ascites and pleural effusion, thrombosis prevention, and treatment for eventual deep thrombosis. Severe OHSS can be fatal, while mild OHSS only causes discomfort that subsides within a few days. When there is a high risk of OHSS, the cycle may be terminated before oocyte retrieval, or all embryos may be cryopreserved for later use in a normal menstrual cycle or a new hormone induction cycle. In this case, the formulation or dosage may be adjusted to mitigate the side effect.

[0005] However, both procedures are subjective; to date, no clinically useful predictive biomarkers have been found for this decision, whether for OHSS cases or for the initial treatment cycle.

[0006] It has long been known that the composition of the hormones used can have an impact. Considering the different preparations used; purified or highly purified urinary preparations (such as human menopausal gonadotropin (hMG)) and recombinant gonadotropins (such as recombinant FSH (rFSH) and biosimilars), clinicians face the question of which to use to obtain enough oocytes for fertilization while avoiding overstimulation.

[0007] For decades, hMG has been considered superior to rFSH in pregnancy rates and live births, while rFSH is superior to hMG in stimulating follicle development. (Pacchiarotti) people, 2010, Fertility and Sterility: 94(6):2467-9; Westergaard people, 2001, Fertility and Sterility: 76(3):543-9)

[0008] Besides the hormones used, changes in the FSH receptor (FSHR) can affect treatment outcomes. Examples include spontaneous OHSS in non-pregnant women (in whom FSHR mutations are found) and familial cases of spontaneous OHSS during pregnancy (in which heterozygosity for FSHR mutations has been identified). Mutations in FSHR are uncommon or even absent in iatrogenic OHSS cases (Kerkelä). people, 2007, Fertil Steril: 87(3):603-6).

[0009] The previously described single nucleotide polymorphism in FSHR is an adenine-to-guanine substitution located in exon 10, which results in an asparagine (N) to serine (S) substitution at position 680 (SNP database code rs6166). Several clinical studies have shown that, despite the same FSH consumption and treatment cycles, homozygous serine (SS) women have fewer oocytes available for fertilization after hormone stimulation compared to women with the same asparagine (NN) codon.

[0010] This polymorphism in FSHR may also be related to male reproductive parameters. One study found that in general males, the NN genotype of male FSHR N680S was associated with high testosterone levels, increased testicular size, increased sperm count, but decreased serum FSH concentration (Lindgren). people, Pharmacogenetics and genomics. 2012;22(9):667-72). FSH treatment was administered to men with idiopathic infertility, but significant differences in response were observed (Santi...). people, 2015, Endocrine connections: 4(3): R46-58).

[0011] In summary, clinicians still face the challenge of choosing the right gonadotropin for each individual patient. This applies to women undergoing controlled ovarian hyperstimulation to obtain sufficient oocytes for fertilization while avoiding overstimulation, as well as to men receiving FSH treatment for infertility.

[0012] Therefore, there is an urgent need for improved methods of individualized gonadotropin therapy to achieve adequate response while avoiding overstimulation and related side effects.

[0013] The inventors have surprisingly discovered a simple method for stratifying patients based on their N680S FSHR genotype for treatment with specific gonadotropins, thereby optimizing assisted reproduction in, for example, the acquisition of oocytes, pregnancy and live birth, and simultaneously reducing the risk of OHSS and avoiding repeated testing of potentially dangerous stimulation cycles. While such methods are generally expected to require extensive equipment and experienced personnel, which are not currently available to all healthcare providers offering assisted reproduction, the inventors have identified a surprisingly rapid, stable, and cost-effective method for individualizing gonadotropin-based treatment. Summary of the Invention

[0014] This invention provides a method, related polynucleotide primers, and kits for stratifying patients for treatment with human menopausal gonadotropin (hMG) or recombinant follicle-stimulating hormone (rFSH). This invention allows for patient stratification based on their N680S genotype for treatment with hMG or rFSH, in which patients receive gonadotropin therapy that will produce a sufficient response and / or avoid overstimulation.

[0015] This is based on the inventors' remarkable determination that the N680S FSHR genotype is directly correlated with responses to hMG and rFSH, and therefore serves as a highly accurate predictor of response to various gonadotropin treatments. In women undergoing controlled ovarian stimulation with assisted reproduction, genotype-based stratified treatment with rFSH or hMG will improve success rates and reduce the risk of serious side effects.

[0016] The first aspect of the present invention provides a method for stratifying subjects for treatment with human menopausal gonadotropin (hMG) or recombinant follicle-stimulating hormone (rFSH), the method comprising or consisting of the following steps: (a) Provide samples from the subjects to be tested; (b) Determine the subject's follicle-stimulating hormone receptor (FSHR) N680S single nucleotide polymorphism (SNP) genotype; and (c) If the genotype is homozygous for the 680S SNP, the subject is stratified for treatment with hMG; and if the genotype is homozygous for the N680 wild type (WT), the subject is stratified for treatment with rFSH.

[0017] The term "stratification of subjects for treatment" includes determining whether a subject is more likely to benefit from hMG or rFSH treatment. Those skilled in the art will understand that the method of the present invention can be used to provide initial stratification for subjects receiving hMG or rFSH treatment, which can then be followed by further clinical studies and medical considerations (such as medical history) to establish stratification for subjects receiving hMG or rFSH treatment or further treatment.

[0018] Human menopausal gonadotropins (hMG or menopausal hormone) are a mixture of gonadotropins extracted from the urine of postmenopausal women (in which FSH is naturally present in high concentrations). Therefore, hMG may contain FSH and luteinizing hormone (LH). For example, hMG may contain FSH and LH in a 1:1 ratio. hMG may also contain other proteinaceous substances, including human chorionic gonadotropin (hCG). In some embodiments, hMG contains FSH and at least one other gonadotropin.

[0019] The term recombinant FSH (rFSH) includes any FSH protein or fragment thereof that is recombinantly (i.e., synthetically) expressed in a host cell or non-human animal. Furthermore, the rFSH protein may be identical to human-produced FSH. Recombinant FSH can be recombinantly produced in bacterial, yeast, animal, or plant cells. Preferably, the rFSH protein is produced in animal cells, such as Chinese hamster ovary (CHO) cells. Unlike hMG, rFSH does not contain any additional proteins.

[0020] In some embodiments, step (c) includes: stratifying the subject for hMG treatment if the genotype is homozygous 680S SNP or heterozygous; and stratifying the subject for rFSH treatment if the genotype is homozygous N680 wild-type (WT).

[0021] In another embodiment, step (c) includes: stratifying the subject for hMG treatment if the genotype is homozygous 680S SNP; and stratifying the subject for rFSH treatment if the genotype is homozygous N680 wild-type (WT) or heterozygous.

[0022] In a preferred embodiment, the method includes or consists of the following steps: (a) Provide samples from the subjects to be tested; (b) Determine the subject's follicle-stimulating hormone receptor (FSHR) N680S single nucleotide polymorphism (SNP) genotype; and (c) If the genotype is homozygous or heterozygous for the 680S SNP, the subject is stratified for treatment with hMG; and if the genotype is homozygous for N680 wild type (WT), the subject is stratified for treatment with rFSH.

[0023] In some embodiments, rFSH contains post-translational modifications different from those of human-generated FSH. Post-translational modifications may include, but are not limited to, glycosylation, phosphorylation, methylation, ubiquitination, SUMOylation, sulfation, acylation, acetylation, alkylation, and / or hydroxylation.

[0024] The terms "sample to be tested" and "sample from subject" include tissue or fluid samples taken from or derived from a subject, wherein the sample comprises endogenous protein and / or nucleic acid molecules and / or carbohydrate fractions. Preferably, the sample to be tested is derived from a human. The term "providing a sample from a subject" includes a sample that has been obtained from the subject prior to performing the claimed method. In other words, step (a) may include providing a sample obtained from the subject, i.e., a sample that has been obtained from the subject.

[0025] In the method of the present invention, the test sample may be a cell, tissue, or fluid sample (or a derivative thereof), comprising or consisting of: blood (graded or ungraded), plasma, plasma cells, serum, tissue cells, saliva, urine, semen, or, equally preferably, proteins or nucleic acids derived from cell or tissue samples. In some embodiments, the sample in step (a) comprises crude tissue obtained from the subject, i.e., without DNA or protein extraction.

[0026] It should be noted that any sample can be used in the method of the present invention as long as it contains protein and / or nucleic acid molecules that can provide the subject's FSHR N680S genotype information.

[0027] In some embodiments, the sample may be collected from the subject via an oral swab, hair sampling, or blood draw. In some embodiments, the sample is an oral swab.

[0028] In some embodiments, the sample provided in step (a) is selected from the group consisting of saliva, oral swabs, ungraded blood, plasma, serum, tissue fluid, semen, and urine. In some embodiments, the sample provided in step (a) is an oral swab or a saliva sample.

[0029] In some embodiments, the nucleic acids or proteins contained in the sample are extracted, purified, or otherwise enriched prior to step (b) of determining the FSHR N680S single nucleotide polymorphism (SNP) genotype of the subject. In a preferred embodiment, the method of the present invention does not require DNA extraction, purification, and / or enrichment steps. In some embodiments, the method of the present invention does not require centrifugation of the sample prior to step (b). In one embodiment, DNA is extracted from the sample by exposure to alkaline conditions. In a preferred embodiment, DNA is extracted from the sample by contacting the sample with a buffer solution with a pH > 8 (such as a buffer solution with pH > 9, pH > 10, or pH > 11). In some embodiments, the buffer solution contains sodium hydroxide (NaOH), potassium hydroxide (KOH), or a hydrate thereof. In some embodiments, DNA is extracted from the sample by heating the sample to a temperature above 40°C (such as above 40°C, above 45°C, above 50°C, above 55°C, or above 60°C). In a preferred embodiment, DNA is extracted from the sample by incubating the sample in a buffer solution containing NaOH at a temperature ranging from about 60°C to about 70°C. In some embodiments, the exposure to alkaline conditions, the contact with buffer solution, and / or the heating are performed for about 1 minute to about 30 minutes, such as about 3 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 7 minutes to about 13 minutes, preferably about 10 minutes. In a preferred embodiment, DNA is extracted from the sample by contacting the sample with a buffer solution containing NaOH at about 65°C for about 10 minutes.

[0030] The term "follicle-stimulating hormone receptor (FSHR) N680S single nucleotide polymorphism (SNP)" refers to the substitution of asparagine (N) for serine (S) at position 680 of the FSHR peptide, as well as any nucleotide substitution in the FSHR gene encoding this substitution. Various nucleotide sequences can encode the FSHR peptide and the N-to-S substitution at position 680, including the adenine (A)-to-guanine (G) substitution defined by the SNP identifier rs6166. The terms N680S, 680S, and rs6166 are used interchangeably and should be understood to have the same meaning as described above, namely, the SNP of asparagine (N) for serine (S) at position 680 of the FSHR peptide.

[0031] The terms “N680 WT,” “680 WT,” or “WT FSHR” are used interchangeably and should be understood as referring to an FSHR polypeptide where position 680 is an asparagine amino acid (i.e., no serine substitution has occurred). The use of the abbreviations “wild-type” or “WT” should not be interpreted as meaning that the FSHR polypeptide or the nucleic acid sequence encoding the FSHR polypeptide necessarily has no other substitutions, deletions, or insertions, but only refers to the identity of position 680 of the polypeptide sequence or the nucleotide sequence encoding that position.

[0032] The terms “N / N,” “N / S,” or “S / S” are also used throughout the text to refer to subjects who have a specific FSHR genotype at position 680. Therefore, “N / N” subjects are homozygous for asparagine (N) at FSHR position 680; “S / S” subjects are homozygous for serine (S) at FSHR position 680; and “N / S” or “S / N” subjects are heterozygous for FSHR position 680, thus carrying one FSHR allele with N at position 680 and one FSHR allele with S at position 680.

[0033] In some embodiments, wild-type FSHR corresponds to the following polypeptide sequence: MALLLVSLLAFLSLGSGCHHRICHCSNRVFLCQESKVTEIPSDLPRNAIELRFVLTKLRVIQKGAFSGFGDLEKIEISQNDVLEVIEADVFSNLPKLHEIRIEKANNLLYINPEAFQNLPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINIHTIERNSFVGLSFESV ILWLNKNGIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVFHGASGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLEKLVALMEASLTYPSHCCAFANWRRQISELHPICNKSILRQEVDYMTQARGQRSSLAEDNESSYSRGFDMTYTEFDYDLCNEVVDVTCS PKPDAFNPCEDIMGYNILRVLIWFISILAITGNIIVLVILTTSQYKLTVPRFLMCNLAFADLCIGIYLLLIASVDIHTKSQYHNYAIDWQTGAGCDAAGFFTVFASELSVYTLTAITLERWHTITHAMQLDCKVQLRHAASVMVMGWIFAFAAALFPIFGISSYMKVSICLPMD IDSPLSQLYVMSLLVLNVLAFVVICGCYIHIYLTVRNPNIVSSSSDTRIAKRMAMLIFTDFLCMAPISFFAISASLKVPLITVSKAKILLVLFHPINSCANPFLYAIFTKNFRRDFFILLSKCGCYEMQAQIYRTETSSTVHNTHPRNGHCSSAPRVTNGSTYILVPLSHLAQN (SEQ ID NO: 1) In certain aspects and embodiments of the method of the present invention herein, any means may be used in step (b) to determine the genotype of the FSHR 680S SNP in the subject.

[0034] In one specific embodiment of the method of the present invention, step (b) includes detecting the presence of a polynucleotide encoding: (i) a 680S SNP of FSHR; (ii) a T307A SNP of FSHR; and / or (iii) an N680 WT.

[0035] The term polynucleotide refers to any molecule that comprises two or more nucleotides or their derivatives.

[0036] The term “FSHR T307A SNP” refers to the mutation (rs6165) in which threonine (T) is replaced by alanine (A) at position 307 of the FSHR polypeptide and / or any nucleotide substitution in the FSHR gene that encodes the replacement.

[0037] Certain genomic loci (including SNP locations) are known to be in linkage disequilibrium. These loci are genetically linked in such a way that they are inherited together during the meiotic division stage of sexual reproduction. The N680S and T307A SNPs of FSHR are known to be in linkage disequilibrium and therefore co-inherited (Wu people, 2017, BMC Med Genet: 18(1):81; Lindgren people, 2012, Parmacogenet Genomics:22(9):667-72)¸ ; rs6166 (SNP) - Populationgenetics - Homo_sapiens - Ensembl Genome Browser 110). Therefore, it is understandable that determining the subject's T307A genotype will also naturally provide the subject's N680S genotype information. vice versa .

[0038] Therefore, in some embodiments of the method of the present invention, the determination of the FSHR 680S SNP genotype in step (b) may include detecting the presence of a polynucleotide encoding the FSHR T307A SNP (i.e., the SNP in which threonine (T) is replaced by alanine (A) at position 307 of FSHR).

[0039] In some embodiments, the polynucleotide is a DNA or RNA molecule. In some embodiments, the polynucleotide is DNA, preferably genomic DNA.

[0040] In some embodiments of the method of the present invention, the presence of polynucleotides is detected using a method selected from the group consisting of: polymerase chain reaction (PCR), allele-specific PCR, loop-mediated isothermal amplification (LAMP), reverse transcription LAMP (RT-LAMP), and reverse transcription PCR (RT-PCR). In situ Hybridization, nanoarrays, microarrays, sequencing, Southern blotting hybridization, or Northern blotting hybridization.

[0041] In some embodiments of the method of the present invention, the detection of the presence of polynucleotides includes PCR or LAMP-based DNA or RNA amplification. The amplicon can then be sequenced, for example by NGS or Sanger sequencing. In some other embodiments, sequencing is not required.

[0042] In some embodiments of this method, the detection of the polynucleotide is performed at a constant temperature. In some embodiments, the DNA amplification of the polynucleotide is performed at a constant temperature.

[0043] In some embodiments of this method, the detection of the polynucleotide is performed at a constant temperature between 20°C-80°C, 30°C-80°C, 40°C-80°C, 45°C-80°C, 50°C-80°C, 60°C-80°C, 20°C-70°C, 30°C-70°C, 40°C-70°C, 45°C-70°C, 50°C-70°C, 55°C-70°C, 60°C-70°C, 20°C-65°C, 30°C-65°C, 40°C-65°C, 45°C-65°C, 55°C-65°C, 55°C-60°C, or 60°C-65°C. For example, the detection of the polynucleotide can be performed at a constant temperature of 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C. In some embodiments, the detection of the polynucleotide can be performed at a constant temperature of 60°C-70°C, such as about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C. In some embodiments, the detection of the polynucleotide is performed at a constant temperature of about 60°C. In some embodiments, the detection of the polynucleotide is performed at a constant temperature of 65°C.

[0044] In some embodiments of the method of the present invention, LAMP is used to detect the presence of the polynucleotide. In a particular embodiment, the LAMP reaction is performed using Bst DNA polymerase.

[0045] In some embodiments of the method of the present invention, the LAMP reaction is carried out at a constant temperature between 20°C-80°C, 30°C-80°C, 40°C-80°C, 45°C-80°C, 50°C-80°C, 60°C-80°C, 20°C-70°C, 30°C-70°C, 40°C-70°C, 45°C-70°C, 50°C-70°C, 55°C-70°C, 60°C-70°C, 20°C-65°C, 30°C-65°C, 40°C-65°C, 45°C-65°C, 50°C-65°C, 55°C-60°C, or 60°C-65°C. For example, the LAMP reaction can be carried out at a constant temperature of 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C. In some embodiments, the LAMP reaction can be carried out at a constant temperature of 60°C–70°C, for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C. In some embodiments, the detection of the polynucleotide is carried out at a constant temperature of about 60°C. In some embodiments, the LAMP reaction is carried out at a constant temperature of 65°C.

[0046] In some embodiments of this method, the LAMP reaction is carried out for approximately 10-20, 10-15, 15-20, 15-25, 15-30, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 20-45, 20-40, 20-35, 20-30, 20-25, 25-30, 25-35, 25-40, 25-45, 30-35, 30-40, 30-45, or 25-30 minutes. In some embodiments of this method, the LAMP reaction is carried out for approximately 25-30 minutes. For example, the LAMP reaction may be carried out for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes. In some embodiments, the LAMP reaction is carried out for 25-30 minutes, for example, 25, 26, 27, 28, 29, or 30 minutes. In some embodiments, the LAMP reaction is carried out for 10-20 minutes, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In some embodiments, the LAMP reaction is carried out for 5-10 minutes, such as 5, 6, 7, 8, 9, or 10 minutes.

[0047] A second aspect of the invention provides a primer set for detecting 680S SNPs and N680 WT FSHRs. Such primers are intended for use in the method of the first aspect of the invention.

[0048] A primer set, in this context, refers to a group of two or more polynucleotide primers. A polynucleotide primer should be understood as any polynucleotide that, alone or in combination with other primers, can instruct DNA or RNA polymerases to initiate DNA or RNA synthesis.

[0049] Exemplary primers that can be used in the method of this invention are listed in Table A: Table A

[0050] The polynucleotide primers of the present invention can be used in various methods for amplifying target polynucleotides. For example, these polynucleotide primers can be used in PCR and / or LAMP-based target polynucleotide amplification. In one embodiment, these polynucleotide primers are used in LAMP-based amplification, in which case they provide a simple, rapid, and cost-effective method. In a preferred embodiment, one or more polynucleotide primers of the present invention are used in step (b) of the first aspect of the present invention described above.

[0051] The 680S SNP is the result of an A-to-G substitution in the DNA encoding FSHR at position 680. To specifically detect the 680S SNP of FSHR, this polynucleotide primer specifically targets the G allele. Correspondingly, to detect N680 WT FSHR, this polynucleotide primer specifically targets the A allele.

[0052] The primers of the present invention may include polynucleotide primers specific to the G allele (FSHR FIP G-specific 1 and FSHR BIP G-specific 1) and polynucleotide primers specific to the A allele (FSHR FIP A-specific 1 and FSHR BIP A-specific 1).

[0053] To reduce off-target amplification, allele-specific primers may contain mismatched nucleotides (i.e., nucleotides that are not complementary to the target sequence). These mismatched nucleotides may be located at the 5'-2 position.

[0054] LAMP amplification typically requires additional outer and loop primers. The primers of this invention include outer primers (FSHR F3 and FSHR B3) and loop primers (FSHR loop F1 and FSHR loop B), which can be used for LAMP-based 680S SNP FSHR or N680WT FSHR amplification.

[0055] Therefore, one aspect of the present invention provides a primer set for detecting 680S SNPs of FSHR, the primer set comprising polynucleotide primers containing the sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0056] In a particular embodiment, the primer set for detecting the 680S SNP of FSHR further includes one or more of the polynucleotide primers containing the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0057] In some embodiments, the primer set for detecting the 680S SNP of FSHR includes polynucleotide primers containing the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0058] Another aspect of the present invention provides a primer set for detecting N680 WT FSHR, comprising polynucleotide primers containing the sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0059] In a particular embodiment, the primer set for detecting N680 WT FSHR further includes one or more of the polynucleotide primers containing the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0060] In some embodiments, the primer set for detecting N680 WT FSHR includes polynucleotide primers containing the sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0061] The primer set of the present invention described herein can be used in step (b) of the method of the present invention to determine the FSHR N680S SNP genotype of a subject.

[0062] Therefore, in some embodiments of the method of the first aspect of the invention, step (b) includes using the primer set defined in the second aspect of the invention.

[0063] Accordingly, in some embodiments of the method of the present invention, step (b) includes detecting the presence of a polynucleotide encoding a 680S SNP of FSHR, and optionally a polynucleotide encoding an N680 WT FSHR. The presence of this polynucleotide was detected using LAMP. The presence of the 680S SNP polynucleotide in FSHR was detected using the first primer set provided herein; and optionally, The presence of N680 WT FSHR was detected using the second primer set provided in this paper.

[0064] In some embodiments, step (b) includes detecting the presence of a polynucleotide encoding the 680S SNP of FSHR and a polynucleotide encoding N680 WT FSHR. The presence of this polynucleotide was detected using LAMP. The presence of the 680S SNP of the FSHR polynucleotide was detected using the first primer set provided in this paper; and The presence of N680 WT FSHR was detected using the second primer set provided in this paper.

[0065] In some embodiments, the first primer set for detecting 680S SNPs of FSHR polynucleotides includes polynucleotide primers containing the sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0066] In additional or alternative embodiments, the first primer set for detecting the 680S SNP of FSHR further includes one or more of the polynucleotide primers containing the sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and / or SEQ ID NO:7.

[0067] In some embodiments, the second primer set for detecting N680 WT FSHR includes polynucleotide primers containing the sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0068] In additional or alternative embodiments, the second primer set for detecting N680 WT FSHR further includes one or more of the polynucleotide primers containing the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

[0069] In additional or alternative embodiments, the first primer set for detecting the 680S SNP of FSHR comprises polynucleotide primers containing the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and the second primer set for detecting N680 WT FSHR comprises polynucleotide primers containing the sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0070] In some embodiments, a first primer set and a first nucleic acid clamp are used to detect the presence of the 680S SNP of the FSHR polynucleotide, the clamp being capable of annealing to the A allele of FSHR and blocking LAMP-based FSHR polynucleotide DNA amplification. In additional or alternative embodiments, a second primer set and a second nucleic acid clamp are used to detect the presence of N680 WT FSHR, the clamp being capable of annealing to the G allele of FSHR and blocking LAMP-based FSHR polynucleotide DNA amplification.

[0071] The term "nucleic acid clamp" refers to any nucleic acid molecule capable of binding to a specific target sequence and blocking the initiation or extension of DNA synthesis of that target sequence. In the method of this invention, nucleic acid clamps may be included in the LAMP reaction mixture to improve the specificity of the genotyping method. Specifically, a nucleic acid clamp capable of annealing to the A allele of FSHR can block non-specific LAMP-based DNA amplification in the sample while still allowing G allele amplification to detect the 680S SNP of FSHR. Similarly, a nucleic acid clamp capable of annealing to the G allele of FSHR can block non-specific LAMP-based DNA amplification in the sample while still allowing A allele amplification to detect N680 WT FSHR. Figure 7 A schematic diagram of the nucleic acid clamp function in detecting N680S SNPs of FSHR is shown, illustrating LAMP-based DNA amplification.

[0072] In some embodiments, the first and / or second nucleic acid clamps comprise or are composed of peptide nucleic acids (PNAs) and / or locked nucleic acids (LNAs). In a preferred embodiment, the first and second nucleic acid clamps comprise or are composed of peptide nucleic acids.

[0073] In a preferred embodiment, the first nucleic acid clamp comprises or is composed of the nucleic acid sequence 5'-CCAATGGTTCCACTTACA-3' (SEQ ID NO: 27), or comprises or is composed of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 27.

[0074] In additional or alternative embodiments, the second nucleic acid clamp comprises or is composed of the nucleic acid sequence 5'-CCAGTGGTTCCACTTACA-3' (SEQ ID NO: 28), or comprises or is composed of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 28.

[0075] In one embodiment, the first primer set is the primer set as defined above. In an additional or alternative embodiment, the second primer set is the primer set as defined above.

[0076] In a preferred embodiment of the method of the present invention, step (b) includes detecting the presence of a polynucleotide encoding a 680S SNP of FSHR and a polynucleotide encoding N680 WT FSHR. The presence of this polynucleotide was detected using LAMP. The presence of the 680S SNP of the FSHR polynucleotide was detected using the first primer set and first nucleic acid clamp provided herein, which anneal to the A allele of FSHR and block LAMP-based FSHR polynucleotide DNA amplification, and optionally... The presence of N680 WT FSHR was detected using the second primer set and second nucleic acid clamp provided in this paper, which annealed with the G allele of FSHR and blocked LAMP-based amplification of FSHR polynucleotide DNA.

[0077] A preferred embodiment of the first aspect of the invention includes the additional step of treating the subject with hMG and / or rFSH according to the stratification of the first aspect of the invention.

[0078] Therefore, a preferred embodiment of the first aspect of the present invention includes the following additional steps: (d) If the subject is homozygous for the 680S SNP of FSHR, administer hMG to the subject; and / or (e) If the subject is homozygous N680 WT, then administer rFSH to the subject.

[0079] In one embodiment, step (d) includes administering hMG to the subject if the subject is homozygous for the 680S SNP of FSHR or heterozygous for N680SFSHR.

[0080] In one embodiment, step (e) includes administering rFSH to the subject if the subject is homozygous for N680 WT or heterozygous for N680S FSHR.

[0081] Additional drug treatments, including gonadotropin-releasing hormone antagonists or agonists, may be administered to subjects before, during, or after the administration of hMG or rFSH according to the first aspect of the invention.

[0082] In some embodiments, subjects treated with rFSH are subsequently treated with hCG.

[0083] In some embodiments, according to the first aspect of the invention, the subject is treated with hMG or rFSH and with luteinizing hormone (LH) and / or human chorionic gonadotropin (hCG).

[0084] In additional or alternative embodiments, the subject may be further treated with a selective estrogen receptor modulator. In some preferred embodiments, the subject may be further treated with clomiphene. In some preferred embodiments, the subject may be further treated with progesterone.

[0085] In a preferred embodiment of the method of any aspect of the present invention, the subject is a human female.

[0086] In some additional or alternative embodiments, the subject is experiencing involuntary childlessness. In a preferred embodiment, the subject is a woman experiencing involuntary childlessness.

[0087] The term "involuntary childlessness" includes situations where the participant is unable to conceive or have children due to medical reasons.

[0088] In some additional or alternative embodiments, the subject suffers from infertility. In a preferred embodiment, the subject is female and infertile. In one embodiment, the woman herself is infertile, i.e., despite having a fertile male partner.

[0089] The terms "infertility" and "infertile" refer to individuals or couples who are unable to achieve a clinical pregnancy after 12 months of unprotected sexual intercourse. The term encompasses both male and female infertility, and further includes both primary and secondary infertility.

[0090] Even if the participants themselves are not infertile, or are unable to conceive or have children for other reasons, they may still experience involuntary childlessness. For example, fertile women may experience involuntary childlessness if their male sexual partners are infertile or unable to conceive or have children for other reasons. Or, fertile men may experience involuntary childlessness if their female sexual partners are infertile or unable to conceive or have children for other reasons.

[0091] Therefore, in some embodiments, subjects may experience involuntary childlessness, but may not be infertile.

[0092] In some additional or alternative embodiments, the subject is receiving or plans to receive controlled ovarian hyperstimulation. In a preferred embodiment, the subject is receiving or plans to receive controlled ovarian hyperstimulation for assisted reproduction.

[0093] "Controlled ovarian hyperstimulation," "ovarian hyperstimulation," and "ovarian stimulation" are used interchangeably in this article and should be understood as any medical procedure involving the use of fertility drugs to induce ovulation of multiple follicles.

[0094] The term "assisted reproduction" encompasses any medical procedure used to address involuntary infertility or childlessness. This procedure may involve retrieving oocytes or sperm from the subject. Such procedures may include... in vitro In vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or gamete / embryo cryopreservation.

[0095] Therefore, in some embodiments of the method of the present invention, the subject is undergoing or planning to undergo controlled ovarian hyperstimulation to obtain oocytes for IVF, ICSI or cryopreservation.

[0096] Therefore, another aspect of the present invention provides a method for treating involuntary childlessness in a subject, the method comprising the following steps: (a) According to the method of the first aspect of the invention, subjects are stratified for treatment with hMG or rFSH; (b) administering hMG or rFSH to the subjects according to the stratification; and (c) Obtaining oocytes from the subject for assisted reproduction.

[0097] In some embodiments, the method is used to treat infertility in a subject.

[0098] In some embodiments, in subjects stratified by the method according to the first aspect of the invention as being treated with hMG or rFSH, the response to hMG and / or rFSH is enhanced.

[0099] In some additional or alternative embodiments, subjects stratified by the method according to the first aspect of the invention to be treated with hMG or rFSH, on average, showed enhanced response to hMG and / or rFSH compared to unstratified subjects.

[0100] The term "response to hMG and / or rFSH" includes any measurable clinical response to treatment, including but not limited to the number of follicles, oocytes retrieved after treatment, pregnancy rate, live birth rate, and baby homecoming rate.

[0101] Therefore, in certain embodiments of any method of the present invention, the number of oocytes obtained from subjects stratified as those treated with hMG or rFSH according to the method of the first aspect of the present invention is increased compared to unstratified subjects.

[0102] In a preferred embodiment, the number of oocytes obtained from subjects stratified as being treated with hMG or rFSH according to the method of the first aspect of the invention is increased compared to unstratified subjects.

[0103] In additional or alternative embodiments, the number of oocytes obtained from subjects stratified by the method according to the first aspect of the invention for treatment with hMG or rFSH is between about 10-20, 12-20, 15-20, 12-17, or 12-15.

[0104] In additional or alternative embodiments, the average number of oocytes obtained from subjects stratified by the method according to the first aspect of the invention for treatment with hMG or rFSH is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% higher than that from unstratified subjects. In some embodiments, the average number of oocytes obtained from subjects stratified by the method according to the first aspect of the invention for treatment with hMG or rFSH is about 40% higher than that from unstratified subjects.

[0105] Another aspect of the present invention provides a method for reducing the risk of ovarian hyperstimulation syndrome (OHSS) in subjects undergoing ovarian stimulation, the method comprising the following steps: (a) According to the method of the first aspect of the invention, subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

[0106] In some embodiments of the method of the present invention, the subject is male.

[0107] FSH has been shown to be essential for normal reproductive function in male subjects, and FSH treatment is considered a promising approach for treating male infertility. In male infertility, the indication for treatment with FSH-containing compositions is to induce and maintain spermatogenesis in patients with hypogonadotropic hypogonadism, although FSH preparations are also used to treat gonadotropin-normal infertile men with idiopathic spermatogenesis disorder (Behçet's disease). , 2019, Front Endocrinol: 10:322; Oduwole people, 2018, Front Endocrinol: 9:763. Homozygous N680 WT FSHR men have been shown to have lower serum FSH concentrations and higher serum testosterone concentrations. Furthermore, higher sperm concentrations and total sperm counts are known in these subjects (Lindgren). people, 2012, Pharmacogenetics and Genomics: 22(9); Grigorova M people, 2013, Andrology: 1(293)).

[0108] The N680S FSHR genotype is associated with responses to hMG and rFSH, and thus serves as a highly accurate predictor of response to treatment with each gonadotropin. This surprising finding is therefore applicable to male patients. The method of the present invention thus allows for the stratification of male subjects based on genotype for treatment with rFSH or hMG.

[0109] Accordingly, another aspect of the present invention provides a method for treating infertility in male subjects, the method comprising the following steps: (a) According to the method of the first aspect of the invention, subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

[0110] In some embodiments, male subjects may have hypogonadotropic hypogonadism.

[0111] In alternative or additional embodiments, the subject may be a male with normal gonadotropin levels but impaired spermatogenesis.

[0112] In some embodiments of any method of the present invention, the method of the first aspect of the present invention stratifies male subjects treated with hMG or rFSH as having increased spermatogenesis, testosterone levels, sperm count, or sperm concentration, compared to unstratified subjects.

[0113] Infertile men and men with declining fertility often have high levels of sperm DNA fragmentation. The sperm DNA fragmentation index (DFI) is considered a predictor of the probability of conception, and FSH treatment is known to improve this parameter in infertile men.

[0114] Therefore, in some embodiments of any method of the present invention, the sperm DNA fragmentation index is reduced in male subjects stratified by the method of the first aspect of the present invention as being treated with hMG or rFSH, compared with unstratified subjects.

[0115] Another aspect of the present invention provides the use of any primer sets and / or nucleic acid clamps described herein in the treatment of infertility including the following steps or in reducing the risk of OHSS in subjects undergoing ovarian stimulation: (a) According to the method of the first aspect of the invention, subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

[0116] In some embodiments, this use includes stratifying subjects for treatment with hMG or rFSH according to a method of the first aspect of the invention, wherein step (b) includes detecting the presence of a polynucleotide encoding a 680S SNP of FSHR and a polynucleotide encoding N680 WTFSHR. The presence of this polynucleotide was detected using LAMP. The presence of the 680S SNP of the FSHR polynucleotide was detected using the first primer set provided herein and optionally the nucleic acid clamp provided herein. The presence of N680 WTFSHR was detected using the second primer set provided herein and optionally the nucleic acid clamp provided herein.

[0117] Another aspect of the invention provides the use of any primer set and / or nucleic acid clamp described herein for stratifying subjects for treatment with hMG or rFSH according to any method of the first aspect of the invention.

[0118] In some preferred embodiments, any of the methods described herein are in in vitro conduct.

[0119] Another aspect of the present invention provides a kit for determining the FSHR N680S SNP genotype of a subject, the kit comprising any of the primer sets and / or nucleic acid clamps described herein.

[0120] In some embodiments, the kit includes a first primer set for detecting FSHR polynucleotide 680S SNP, the first primer set including polynucleotide primers containing the sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0121] In additional or alternative embodiments, the kit includes a primer set for detecting N680 WT FSHR, the primer set comprising polynucleotide primers containing the sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0122] In additional or alternative embodiments, the kit includes a first primer set for detecting 680S SNPs of FSHR, the first primer set including polynucleotide primers containing the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and a second primer set for detecting N680 WT FSHR, the second primer set including polynucleotide primers containing the sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0123] In some embodiments, the kit includes a first nucleic acid clamp for detecting FSHR polynucleotides in a 680S SNP, the first nucleic acid clamp containing the sequence of SEQ ID NO: 27.

[0124] In additional or alternative embodiments, the kit includes a second nucleic acid clamp for detecting N680 WT FSHR polynucleotides, the second nucleic acid clamp containing the sequence of SEQ ID NO: 28.

[0125] In some embodiments, the kit further includes a DNA polymerase. In a preferred embodiment, the DNA polymerase is included in the master mixture. In some embodiments, the kit includes a DNA polymerase. For example, the DNA polymerase may be selected from Bst or Bsm DNA polymerase.

[0126] It should be understood that PCR or LAMP-based target sequence amplification can be detected or visualized using a variety of detection systems, such as gel electrophoresis, colorimetric indicators, turbidity measurements, and dye-binding methods. Amplification can also be detected based on changes in solution pH during DNA synthesis, which cause color changes.

[0127] Accordingly, in some embodiments, the kit further includes a pH indicator. In a preferred embodiment, the kit further includes phenol red.

[0128] In alternative or additional embodiments, the kit further includes a DNA-binding dye. In a preferred embodiment, the kit further includes SYBR Green.

[0129] In a preferred embodiment, the kit includes: (i) Primer sets as disclosed herein; (ii) DNA polymerase, optionally included in the master mixture; (iii) Phenol red; and (iv) (Optional) DNA lysis buffer.

[0130] In a preferred embodiment, the kit also includes the nucleic acid clamp disclosed herein.

[0131] In one embodiment, the DNA lysis buffer comprises sodium hydroxide (NaOH) or potassium hydroxide (KOH). In some embodiments, the DNA lysis buffer has a pH >8, such as a buffer with pH >9, a buffer with pH >10, or a buffer with pH >11.

[0132] In some embodiments, the kit described herein is used in the method of the first aspect of the invention.

[0133] Preferred, non-limiting examples embodying certain aspects of the invention will now be described with reference to the following accompanying drawings: Figure 1 ：A schematic diagram illustrating the impact of patient stratification according to the method of the present invention on pregnancy and live birth in women undergoing assisted reproduction. Pregnancy and live birth rates in a Swedish clinic. (1) Study participants receiving hormone therapy based on genotypes in FSHR N680S determined by the method of the present invention (n=221). (2) Unclassified population matched for age, BMI, oocyte reserve, and inclusion / exclusion criteria (n=991).

[0134] Figure 2 Schematic diagram of the genotyping method. Samples are collected from subjects and DNA is extracted. The extracted DNA is used as a template in a LAMP reaction using G-specific primers for allele-specific amplification of N680S FSHR. DNA amplification in N680SFSHR positive samples results in a color change from pink to yellow (shown in the diagram as a change from dark gray to light gray). Positive signals are observed in the DNA of homozygous S / S or heterozygous N / S genotype subjects.

[0135] Figure 3: LAMP was performed for 25 minutes at a temperature range of 62-68°C. 50 ng of DNA extracted from blood was used as a template. Figure 3a The results were indicated by the presence of the pH indicator phenol red. In tubes with a positive reaction, the color changed from pink to yellow (from dark gray to light gray) after DNA amplification. A positive signal was observed in reactions containing DNA from homozygous S / S genotypes and heterozygous N / S genotypes. Figure 3a -b).

[0136] Figure 4 ： Representative images from N680S DNA Sanger sequencing. DNA sequence from a homozygous A subject; genotype: N / N. DNA sequence from a heterozygous A / G subject; genotype: N / S. DNA sequence from a homozygous G subject; genotype: S / S.

[0137] Figure 5 ： This diagram illustrates the binding sites of the LAMP primers and nucleic acid clamps (PNAs) of this invention at the FSHR locus. The G allele and A allele (680S and N680 WT FSHR, respectively) are presented in an optional format (G / A).

[0138] Figure 6 ： An improved LAMP-based genotyping method (see Example 4) and a schematic diagram of patient stratification according to the method of the present invention. The schematic diagram illustrates a G-specific LAMP assay for detecting 680s SNP FSHR.

[0139] Figure 7 ：A schematic diagram illustrating the function of a PNA clamp. Specifically, a PNA clamp that anneal to the A allele of FSHR can block non-specific LAMP-based DNA amplification in the sample while still allowing G allele amplification to detect the 680S SNP of FSHR. Similarly, a PNA clamp that anneal to the G allele of FSHR can block non-specific LAMP-based DNA amplification in the sample while still allowing A allele amplification to detect N680 WT FSHR. The diagram illustrates an A-specific PNA clamp.

[0140] Figure 8 ： A schematic diagram (top) and a representative image (bottom) of the LAMP-based genotyping method described in Example 4.

[0141] Figure 9 ： A schematic diagram (top) and a representative image (bottom) of the sensitivity limit of the LAMP-based genotyping method described in Example 4.

[0142] Example 1

[0143] introduction

[0144] Assisted reproduction is widely used to treat infertility and involuntary childlessness. It is estimated that more than 2.5 million hormonal cycles are completed globally each year, and approximately 500,000 babies are born annually after assisted reproduction. To date, approximately 10 million children have been born after assisted reproduction, and this number is expected to continue to grow in the coming years due to delayed childbearing and easier access to effective assisted reproductive technologies, including in developing countries (1-4).

[0145] The most commonly used fertilization technique is intracytoplasmic sperm injection (ICSI), which involves injecting a single sperm into eggs retrieved from a female partner, followed by selection of the best embryos for transfer. Overall, ICSI accounts for approximately 65% ​​of all treatments worldwide, while conventional in vitro fertilization (IVF), in which oocytes are retrieved through natural fertilization, constitutes the remaining 35% (5). These percentages vary significantly between countries, but regardless of the technique used, female partners initially undergo so-called controlled ovarian hyperstimulation, achieved through the gonadotropin follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) agonist human chorionic gonadotropin (hCG), which releases multiple follicles and subsequently multiple oocytes for maturation.

[0146] The optimal number of mature follicles ready to release oocytes is 10-15, but the range can be from zero to over 20. A number exceeding 20 is defined as ovarian hyperstimulation syndrome (OHSS), the most concerning iatrogenic side effect of this therapy. In addition to polyfollicular ovaries, severe OHSS is characterized by enlarged ovarian volume, abdominal pain, increased vascular permeability, and leakage of intracellular fluid into the extracellular space, leading to hemoconcentration and an increased risk of thrombosis. These women require medical intervention including parenteral fluid administration, drainage of ascites and pleural effusion, thrombosis prevention, and treatment for eventual deep thrombosis. Severe OHSS can be fatal, while mild OHSS only causes discomfort that subsides within a few days. When there is a high risk of OHSS, the cycle may be terminated before oocyte retrieval, or all embryos may be cryopreserved for later use in a normal menstrual cycle or a new hormone induction cycle. In this case, the formulation or dosage may be adjusted to mitigate the side effect.

[0147] However, both procedures are subjective; to date, no clinically useful predictive biomarkers have been found for this decision, whether for OHSS cases or for the initial treatment cycle.

[0148] It has long been known that the composition of the hormones used can have an impact. Considering the different preparations used; purified or highly purified urinary preparations (such as menopausal gonadotropins (hMG)) and recombinant gonadotropins (such as recombinant FSH (rFSH) and biosimilars), clinicians face the question of which to use to obtain enough oocytes for fertilization while avoiding overstimulation.

[0149] For decades, hMG has been considered superior to rFSH in terms of pregnancy rate and live birth rate, while rFSH is superior to hMG in terms of stimulating follicle production (6).

[0150] In addition to the hormones used, changes in the FSH receptor (FSHR) and LHCGR (which mediates the effects of both LH and hCG) can affect treatment outcomes. Examples of this include spontaneous OHSS in non-pregnant women (in whom FSHR mutations were found) (7, 8), and familial cases of spontaneous OHSS during pregnancy (in which heterozygosity to FSHR mutations has been identified) (8-11). In iatrogenic OHSS cases, mutations in FSHR are uncommon or even absent (12).

[0151] The previously described single nucleotide polymorphism in FSHR is an adenine-to-guanine substitution located in exon 10, which results in a substitution of amino acid 680 asparagine (N) for serine (S) (SNP database code rs6166). Several clinical studies have shown that, despite the same FSH consumption and treatment cycles, homozygous serine (SS) women have fewer oocytes available for fertilization after hormone stimulation compared to women with the same codon asparagine (NN) (13).

[0152] Therefore, stratified treatment based on medication and genotype may be an optimal option for assisted reproduction in terms of oocyte retrieval, pregnancy rates, live births, and avoiding the potential risks of repeated testing stimulation cycles. To investigate this, over 800 women from two clinical units using different fertilization methods were consecutively randomized to controlled ovarian stimulation with hMG or rFSH and genotyped. The primary outcomes were oocyte count, pregnancy, live birth, and adverse events.

[0153] Materials and methods

[0154] Research Design

[0155] Prior power calculations were performed with a sample size of 810 randomized subjects per treatment group to obtain ≥80% efficacy and a two-sided significance level of 0.05. Inclusion criteria were: unprotected sex for >12 months, age <40 years, bilateral ovarian reserve, and body mass index (BMI) <30 kg / m². 2Normal ovulation cycles (26-28 days), and infertility due to tubal factors, male factors, or unexplained infertility were considered as indications for treatment. Exclusion criteria included: anti-Müllerian hormone (AMH) <5 pmol / L or FSH >12 IU / L on day 2-3 of the cycle, endometriosis, polycystic ovary syndrome, premature ovarian failure, smoking, or male age >56 years. From September 2016 to December 2020, a total of 948 women were evaluated for eligibility. Of these, 535 were recruited from Malmö, Sweden, and 413 from Poznan, Poland. A total of 810 women were ultimately enrolled, including 475 Swedish women and 335 Polish women. At initial consultation, Swedish women were randomized via sealed envelopes (concealing the allocation protocol from participants and healthcare staff) to receive controlled ovarian stimulation with rFSH (Gonal-f®, Merck Serono SA Aubonne, Switzerland) or hMG (Menopur®, Ferring Pharmaceuticals, St:Prex, Switzerland). In Poland, simple randomization was used. Venous blood samples were collected for hormone testing and DNA extraction. Following this, standard clinical procedures were performed. The primary outcome was the number of oocytes aspirated (≥15). Secondary outcomes included the number of high-quality embryos (GQE), pregnancy, live birth, and adverse events, namely miscarriage (for fresh embryo transfer only) and OHSS.

[0156] patient characteristics

[0157] There were no differences between Swedish and Polish women in terms of age, total ovarian reserve (GQE) measured by anti-Müllerian hormone (AMH), and gQE; only slight differences were found in BMI and the number of oocytes aspirated (Table 1). A total of 438 women (54%) were treated with rFSH, while the remainder (372, 46%) received hMG. Women stimulated with hMG had higher total hormone doses compared to those treated with rFSH. Polish women had higher OSI (out-of-body mass index).

[0158] treat

[0159] In Poland, 81% of cases used ICSI, while in Sweden, conventional IVF was the predominant method (52%). 98% of cases used GnRH antagonist protocols (Ganirelix®, Orgalutran, Organon [Sweden] Ltd, Stockholm, Sweden, or Fyremadel, SUN Pharmaceutical Industries Europe BV, Hoofddorp, Netherlands). Twenty women (2%) used a standard long-term protocol using GnRH agonists (Synarela®, Nafarelin, Pfizer AB, Sollentuna, Sweden). Controlled ovarian stimulation was achieved through daily, individually administered doses of hMG or rFSH. Follicular development was monitored via transvaginal ultrasound on days 6–8 of stimulation, with individual dose adjustments as needed. When three or more follicles reached 18 mm, 250 mcg hCG (Ovitrelle, Merck KGaA, Darmstadt, Germany) was administered to induce follicular maturation and oocyte release. Oocyte aspiration was performed 36 hours later. For luteal support, progesterone (Lutinus, Ferring SA Hilding, Lausanne, Switzerland) was administered vaginally at 3 x 200 mg / day for 14 days. Simultaneously, oocyte fertilization was performed, and the embryos were maintained in an incubator until the luteal phase, during which the optimal embryo was transferred to the uterus. Single embryo transfer was used only. A pregnancy test (serum hCG) was performed 14 days later. Pregnancy was confirmed by ultrasound at 6 weeks of gestation.

[0160] All participating women provided written informed consent. Ethical approval was obtained from the Swedish Ethics Review Board (2016-467) and the corresponding Polish institution. The study was also reported to ClinTrials.Gov (NCT 03737253).

[0161] Genotyping

[0162] DNA was extracted from peripheral leukocytes using the PureLink™ Genomic DNA Miniprep Kit (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). The FSHR N680S and LHCGR N312S variants were analyzed by TaqMan allele identification in a 25µl reaction system containing 10 ng g DNA on a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Stockholm, Sweden) (FSHR: detection ID C_2676874_10_; rs6166; LHCGR: detection ID C_460917_1_; rs2293275, probes with FAM™ and VICR dyes, Life Technologies, Carlsbad, CA, USA). Genotyping was performed at the Centre for Translational Genomics, Lund University (CTG; Faculty of Medicine, Lund University, Lund, Sweden). Randomly selected samples were directly sequenced using Sanger sequencing (LightRun sequencing, GATC Services, Eurofins Genomics, Ebersberg, Germany) to verify genotypes.

[0163] statistics

[0164] The allele frequencies of FSHR N680S and LHCGR N312S polymorphisms were analyzed using the chi-square test, compared with a control group (Kuijper et al., 2010(17), for FSHR N680S; and Piersma et al., 2007(18), for LHCGR N312S). All residuals were tested for normality using the Kolmogorov-Smirnov test. For all women and different combinations of FSHR N680S and LHCGR N312S genotypes, clinical outcomes and hormone preparations used before assisted reproduction were compared using the chi-square test, one-way ANOVA, or Mann-Whitney U test (as appropriate).

[0165] This study was conducted on candidate genes, therefore no correction was made for multiple comparisons. Furthermore, randomization ensured a good balance between known and unknown covariates on average across treatment groups, and no adjustments were made in the analysis. Data were analyzed using SPSS version 28 (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.

[0166] result

[0167] Aspirated oocytes

[0168] The mean number of oocytes retrieved in subjects treated with hMG and rFSH was 9.8 and 10.3, respectively. This was also reflected in the number of high-quality embryos (1.7 vs 1.9). In women with homozygous asparagine (NN) N680S FSHRs, significantly more oocytes were retrieved when treated with rFSH instead of hMG (rFSH 10.0 ± 7.3 vs shMG 8.0 ± 5.0, p = 0.002, Table 3A). This same result was evident regardless of location (Sweden: rFSH 12.0 ± 8.0 vs hMG 9.0 ± 6.0, p = 0.003; Table 3B; Poland: rFSH 9.0 ± 6.0 vs hMG 7.0 ± 5.0, p = 0.010, Table 3C). Compared to NN or heterozygous (NS) women, homozygous serine (SS) carriers had a significantly higher chance of obtaining ≥15 oocytes in response to hMG (SS 33%, NS 14%, NN 14%, p<0.001, Table 4). On the other hand, NN women responded best to rFSH (NN 25%, NS 15%, SS 15%, p=0.061, Table 4). As for LHCGR N312S, there was clearly no difference in the chance of developing ≥15 oocytes between genotypes (rFSH p=0.707, hMG p=0.679).

[0169] Pregnancy and live birth

[0170] A total of 426 pregnancies were achieved from the first cycle; 260 were after fresh embryo transfer and 166 after frozen embryo transfer. Some women (n=28) received both fresh and frozen embryo transfer (16 hMG, 12 rFSH treatment), resulting in 398 clinical pregnancies; 193 (52%) and 205 (47%) were treated with hMG and rFSH, respectively. Overall, a statistically significant hormone-dependent difference of 11% was observed in pregnancy rates after fresh embryo transfer (hMG 38% vs. rFSH 27%, p=0.002). The pregnancy rate was even higher after fresh transfer in the Swedish cohort (hMG 43% vs. rFSH 33%; p=0.025), while this difference was not observed in the Polish cohort. With frozen embryo transfer, hormone type and fertilization method had no effect (ICSI 30% vs. IVF 37% pregnancy rate, p=0.184).

[0171] A total of 331 live births were recorded in the first cycle, representing 78% of all pregnancies. Five of these were twin deliveries, and one was a pair of siblings—originating from frozen embryos from the first cycle. Following fresh embryo transfer, the live birth rate was 9% higher with hMG than with rFSH (hMG 28% vs rFSH 19%, p=0.003). In the Swedish cohort, an 11% higher live birth rate was observed with hMG compared to rFSH (hMG 34% vs rFSH 23%, p=0.009). No such differences were recorded with frozen embryos.

[0172] Relationship between pregnancy and live birth and genotype

[0173] The distribution of gonadotropin receptor genotypes in both countries and the overall population was consistent with the expected proportions (Table 2). The allele frequencies of FSHR N680S were 56% N and 44% S (17, 18). p =0.776), for LHCGR N312S, 45% N and 55% S, which was no different from previous reports (p=0.474)(18).

[0174] In women receiving fresh embryo transfer and with a FSHR N680S score of SS, controlled ovarian hyperstimulation with hMG was associated with a 15% higher pregnancy rate compared to rFSH (hMG 41% vs. rFSH 26%, p=0.050). These women also had a 17% higher live birth rate (hMG 33% vs. rFSH 16%, p=0.016). Correspondingly, Swedish women responding to hMG had a 22% higher pregnancy rate (hMG 48% vs. rFSH 26%, p=0.021) and a 23% higher live birth rate (hMG 40% vs. rFSH 17%, p=0.010). Heterozygous women treated with hMG also had a 10% higher pregnancy rate than those treated with rFSH (hMG 41% vs. rFSH 31%, p=0.049), but no difference in live birth rate was observed. If frozen embryos are used, the FSHR N680S genotype has no effect. For LHCGR N312S, no association was found with pregnancy or live birth.

[0175] Adverse events

[0176] There was no statistically significant difference in miscarriage rates between the two groups (Sweden 24% vs. Poland 31%, p=0.213), neither in relation to the hormones used for ovarian stimulation (hMG 19% vs. rFSH 32%, p=0.282) nor in relation to genotype (FSHR N680S: NN 30%, NS 25%, SS 24%, p=0.706 and LHCGR N312S: NN 26%, NS 27%, SS 26%, p=0.984). A total of 3 women (0.37%) developed severe OHSS, including those requiring hospitalization. This incidence was not different based on FSHR genotype (p=0.071), LHCGR genotype (p=0.131), or the type of gonadotropin used (p=0.060).

[0177] discuss

[0178] For decades, hMG has been generally considered superior to rFSH in terms of pregnancy rates and live births, while rFSH is superior to hMG in stimulating follicle development, a finding also present in this work. However, for the first time, the inventors have demonstrated through a randomized prospective study that the combination of the FSHR N680S variant with the hormones used also has an effect in this context.

[0179] Women homozygous for serine benefited from hMG treatment, noting a doubling of the chance of producing multiple fertile oocytes compared to women with the same gene variant but treated with rFSH. However, the opposite was observed in women with the same locus, NN. These women, treated with rFSH instead of hMG, had approximately a 40% higher chance of producing 15 or more oocytes. For heterozygous women (NS), hormone type was less important. This finding is consistent with data from a previous retrospective study: 191 women underwent two treatments, using highly purified FSH (HP-FSH) in both cycles, using rFSH in both cycles, or using rFSH in the first cycle followed by HP-FSH in the second cycle (14). For the SS genotype, more oocytes were produced in the HP-FSH cycle; while for the NS genotype, more oocytes were aspirated in the rFSH cycle. No difference was found for the NN genotype, possibly due to insufficient statistical power in this study; only 28 women were homozygous NN. Although HP-FSH cannot be considered equivalent to hMG because the amount of LH / hCG in HP-FSH is much lower (15-17), both preparations are derived from urine, which may affect biological aspects such as cofactor content and clearance, which may differ from rFSH (18).

[0180] The use of frozen or fresh embryos had little impact on the results in this study and was not related to FSHR genotype or hormones. It was also not related to the method of fertilization (ICSI or IVF) or the risk of OHSS that occurred and was clinically treated.

[0181] As a treatment strategy, individualized and therefore more effective treatment will be entirely new for infertility. Currently, no clinics use genetic testing before assisted reproductive technology, but this method can be easily incorporated into routine clinical practice. Improved treatment precision will bring economic benefits to society by reducing repeated, expensive treatment attempts; and it will reduce the physical and psychological suffering of infertile couples. For women who avoid life-threatening side effects, the advantages are naturally immeasurable.

[0182] Table 1. Patient characteristics.

[0183]

[0184] Table 2. Genotype distribution and treatment.

[0185]

[0186] Table 3A. Aspirated oocytes, FSHR genotypes, and hormone reagents.

[0187]

[0188] Table 3B. Aspirated oocytes, FSHR genotypes, and hormone reagents (Sweden).

[0189]

[0190] Table 3C. Aspirated oocytes, FSHR genotypes, and hormone reagents (Poland).

[0191]

[0192] Table 4.15 or more aspirated oocytes, FSHR N680S genotype and hormone reagents.

[0193]

[0194] Example 2

[0195] introduction

[0196] The study population data presented in Example 1 were also analyzed by comparison with a new control cohort (Swedish women, n=911). These women not yetGenotyping was performed, but the population was otherwise matched to the study population and followed standard clinical practice in Swedish IVF clinics at the time. The aim was to compare pregnancy and live birth rates with the group that had not undergone genotyping.

[0197] In this additional study, all analyses were based on the FSHR N680S genotyping. The study population in Example 1 consisted of two cohorts from Sweden and Poland. In this study, these were analyzed as a single cohort (genotyping group). All pregnancies (from fresh and frozen embryos) were pooled for analysis (in Example 1, they were analyzed as independent groups).

[0198] The results showed that a genotype-guided approach could achieve a significant increase in pregnancy and live birth, in which human menopausal gonadotropin (hMG) was administered to FSHR N680S S carriers and recombinant FSH (rFSH) was administered to women carrying NN.

[0199] Materials and methods

[0200] Subjects

[0201] Genotyping cohort

[0202] Inclusion criteria were: unprotected sex for more than 12 months, age less than 40 years, bilateral ovarian reserve, and body mass index (BMI) less than 30 kg / m². 2 Normal ovulation cycles (26-32 days), and infertility due to tubal factors, male factors, or unexplained causes were considered indications for treatment. Exclusion criteria included: serum anti-Müllerian hormone (AMH) levels <5 pmol / L or FSH >12 IU / L on day 2-3 of the cycle, endometriosis, polycystic ovary syndrome (PCOS), premature ovarian failure, smoking, or male age >56 years. From September 2016 to December 2020, a total of 810 female subjects were enrolled and followed up until December 2021. Of these, n=475 were recruited from the Reproductive Medicine Center at Malmö Skåne University Hospital, Sweden, and n=335 were recruited from the Department of Infertility Diagnosis and Treatment at the Medical University of Poznan, Poland. Patient characteristics are listed in Table 1 (see Example 1). At initial consultation, participants were assigned 1:1 to either rFSH (Gonal-f®, Merck Serono SA Aubonne, Switzerland) or hMG (Menopur®, Ferring Pharmaceuticals, St: Prex, Switzerland). Venous blood samples were collected for DNA extraction and then processed according to standard clinical procedures. Only the results of the first COH cycle and corresponding embryo transfer (fresh and frozen) are included. The primary outcome was live birth. Secondary outcomes included pregnancy, number of oocytes aspirated, miscarriage, and OHSS.

[0203] Ungenotyped cohorts

[0204] A total of 991 Swedish women were included who were neither asked to participate in genotyping nor refused it. These women were matched with those identified in the genotyping cohort based on inclusion criteria and time periods. Similarly, among these participants, only the first COH cycle was included, with live birth as the outcome.

[0205] Controlled ovarian hyperstimulation

[0206] In the study population, 437 patients (54%) were treated with rFSH, and 373 patients (46%) received hMG. 98% of subjects used GnRH antagonist regimens (Ganirelix®, Orgalutran, Organon [Sweden] Ltd, Stockholm, Sweden, or Fyremadel, SUN Pharmaceutical Industries Europe BV, Hoofddorp, Netherlands), while 2% used GnRH agonists (Synarela®, Nafarelin, Pfizer AB, Sollentuna, Sweden). Follicular development was monitored by vaginal ultrasound on days 6–8 of stimulation, and individual dosages were adjusted as needed.

[0207] Compared with rFSH treatment, women stimulated with hMG had a higher median (range) total hormone dose (1875.0 [250.0; 6750.0] vs. 1650.0 [31.0; 4875.0], Table 1 (see Example 1)). Follicle maturation and oocyte release were induced when ≥3 follicles reached 18 mm using 250 mcg hCG (Ovitrelle, Merck KGaA, Darmstadt, Germany). Oocytes were retrieved 36 hours later. For luteal support, progesterone (Lutinus, Ferring SAHilding, Lausanne, Switzerland) was administered vaginally at 3 × 200 mg / day for 14 days. Oocyte fertilization was performed concurrently via ICSI or standard IVF (Table 1 (see Example 1)). Optimal embryos were transferred to the uterus, and pregnancy was detected by hCG testing 14 days later and confirmed by ultrasound at 7–8 weeks.

[0208] Miscarriage is defined as the spontaneous loss of a prior pregnancy, confirmed by hCG testing, at any gestational week.

[0209] Genotyping

[0210] DNA was extracted from peripheral leukocytes using the PureLink™ Genomic DNA Miniprep Kit (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) or the Quick-DNA™ Miniprep Kit (Zymo Research Corporation, Irvine, CA, USA). The FSHR N680S and LHCGR N312S variants were analyzed by TaqMan allele identification in a 25 µl reaction volume containing 10 ng of DNA on a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Stockholm, Sweden) (FSHR: detection ID C_2676874_10_; rs6166; LHCGR: detection ID C_460917_1_; rs2293275, probes with FAM™ and VIC dyes, Life Technologies, Carlsbad, CA, USA). Genotyping was performed at the Center for Translational Genomics, Lund University (CTG; Faculty of Medicine, Lund University, Lund, Sweden). Randomly selected samples were directly sequenced using Sanger sequencing (LightRun sequencing, GATC Services, Eurofins Genomics, Ebersberg, Germany) to verify genotypes.

[0211] statistics

[0212] Statistical analyses were performed using R software (version 4.2.2) (R Core Team, 2022) and SPSS software version 29 (SPSS, Inc., Chicago, IL, USA). Background data were described using the mean and standard deviation for numerical variables, and the median and range if non-normally distributed; absolute values ​​and relative frequencies were used for categorical variables. In the genotyping cohort, logistic regression analysis was used to explore the odds ratio (OR) of outcomes after hMG use, with rFSH as a reference, for binary outcomes (pregnancy and live birth per initiated hormonal cycle [both fresh and frozen oocytes]. These calculations were performed separately for FSHR and LHCGR genotypes, and for S allele carriers and non-carriers. Adjustments were made for female age (a continuous variable) and location (Sweden and Poland). Additional adjustments were made for fertilization method (IVF / ICSI) as a sensitivity analysis. For the numerical outcome of oocyte count, linear regression analysis was used, with the outcome transformed by the square root to satisfy the normality assumption. The estimated marginal mean (EMM) of the numerical outcomes was also calculated.

[0213] Chi-square test was used to compare the risks of miscarriage and OHSS. Differences in pregnancy and live birth rates were found between the Swedish subcohort, which underwent genotyping using the same method and received optimal treatment, and those who did not undergo genotyping. Chi-square test was used to assess the allele frequencies of FSHR N680S and LHCGR N312S polymorphisms compared to other populations.

[0214] result

[0215] Background characteristics of genotyping and non-genotyping cohorts

[0216] When considering mean (SD) age (32 years [3.9] vs. 33 years [3.8] and BMI 24 [3.1] vs. 24 [3.4]) or median (range) AMH concentration (19 pmol / L [5.0; 104] vs. 19 pmol / L [6.0; 81.0]), there was no difference between ungenotyped and genotyped women. The same was true for the choice of fertilization method (IVF: 52% vs. 53%, ICSI: 45% vs. 42%, microcombination [50% of oocytes fertilized by IVF, 50% by ICSI]: 2% vs. 6%).

[0217] Correlation between the number of pregnancies and live births and hormone type and genotype

[0218] The distribution of gonadotropin receptor genotypes was consistent with the expected proportions in both study sites and the overall population (19-20) (Table 2, see Example 1). In N680S S carriers (NS / SS), hMG treatment resulted in an 8% higher pregnancy rate than rFSH treatment (54% vs 46%; adjusted OR: 1.41, 95% CI 1.01–1.97, p = 0.046; Table 5). A 5% higher live birth rate was also observed in hMG-treated S carriers, but this difference was not statistically significant (44% vs. 39%, OR: 1.22, 95% CI 0.87–1.71, p = 0.26). In NN carriers, there was no difference in pregnancy or live birth rates between hormones. The same was true for the LHCGR genotype. These results remained unchanged after adjusting for fertilization method.

[0219] Clinical outcomes of women in Sweden who were genotyped and received optimal treatment versus those who were not genotyped.

[0220] Compared with women who were not genotyped (whose hormone selection was based on standard clinical criteria), hMG-stimulated S carriers and rFSH-stimulated NN carriers (i.e., the correct hormones according to genotype) had an 11% higher pregnancy rate in the first cycle (including all transfers) (51% vs 40%, p=0.002). Figure 1 The subsequent live birth rate was also 11% higher (40% vs 29%, p=0.001).

[0221] Aspirated oocytes

[0222] The median number of oocytes retrieved was 9 (Table 1 (see Example 1)). More oocytes were retrieved from N680S NN women (28% vs. 20%, respectively) when treated with rFSH instead of hMG (EMM: 10 vs. 8 aspirated oocytes; β: -0.33, 95% CI -0.55; -0.11, p=0.003, Table 6). This result was independent of study location (EMM: Sweden: 9 aspirated oocytes from hMG vs. 12 from rFSH [more than 25%]; β: -0.44, 95% CI -0.75; -0.14, p=0.004; Poland: 7 oocytes from hMG vs. 9 after rFSH [more than 22%]; β: -0.38, 95% CI -0.68; -0.08, p=0.013). No results were observed regarding LHCGR and the number of oocytes aspirated.

[0223] Adverse events

[0224] Notably, there was no statistically significant difference in the rate of hormone-related miscarriage among study participants (hMG 25% vs. rFSH 28%, p=0.647). When comparing hMG and rFSH, the odds ratio (OR) for miscarriage was not significantly different between women who achieved pregnancy, whether in N680S N homozygotes (OR=0.59; 95% CI: 0.23–1.53; p=0.28) or S allele carriers (OR=1.24; 95% CI 0.65–2.38; p=0.51). The rate of miscarriage was not significantly different in the Swedish optimal treatment subcohort compared to the control group (8% vs. 10%, p=0.243). Three women (0.4%) (all N680S NN and receiving hMG treatment) developed severe OHSS, including those requiring hospitalization. NN was associated with OHSS (p=0.032), but not with hormone type (hMG vs. rFSH) (p=0.060). The total hormone dose administered to women with OHSS was no higher than that to women without OHSS (p=0.917) or to all other stimuli (p=0.839). LHCGR N312S was not associated with OHSS (p=0.799) or miscarriage risk (p=0.761).

[0225] discuss

[0226] The primary concern for couples seeking assisted reproductive technology (COH) is to achieve pregnancy as quickly as possible and ultimately have a live birth. To this end, women are prepared to undergo supraphysiological doses of hormone therapy, despite the potential for negative side effects. Therefore, increasing the chances of pregnancy and live birth in each initiated COH cycle is central. According to a recently published systematic review, a 1% improvement annually results in thousands of additional lives worldwide.

[0227] In this large clinical study, we found that for FSHR N680S carriers, the pregnancy rate was significantly higher for each initiated first COH response hMG compared to rFSH. Even greater clinical significance was observed when comparing a cohort of women receiving the “correct” hormone based on genotype to a matched inclusion / exclusion criteria group of women who were not genotyped (and therefore received the hormone type according to the standard clinical protocol). Among women receiving hormones determined by their FSHR genotype, the chance of giving birth within the first treatment cycle after all transplants (both fresh and frozen) was 40%, a highly statistically different rate from the 29% observed in ungenotyped control women receiving the standard clinical protocol.

[0228] For patients with the NN genotype, pregnancy and live birth rates were not related to the type of hormone administered. However, the number of oocytes aspirated was 33% higher in patients receiving rFSH, consistent with previous meta-analyses on this topic (13). Although the NN variant is considered more sensitive to rFSH than the NS / SS variant, the underlying biological mechanisms remain unclear. This phenomenon is not only observed in... in vitro Observed (21), in vivo This is also true (22-23). ​​It was also confirmed that human granulocytes in N patients homozygous for N680S were more sensitive to rFSH compared to those homozygous for S patients (21).

[0229] Neither the risk of miscarriage nor the risk of OHSS were associated with combinations of N680S FSHR genotype and FSH type. However, this study may not be able to draw any conclusions about these outcomes due to insufficient statistical power. Furthermore, no differences were observed in the LHCGR N312S genotype among any of the included outcomes.

[0230] To the best of our knowledge, this is the largest study conducted to investigate FSHR N680S polymorphism and hormone type (hMG vs. rFSH) in relation to IVF outcomes. This represents a new approach and may mark a first step towards precision medicine in the IVF field. Comparing a genotyped cohort that received FSH as recommended above with an ungenotyped group that selected hormones based on traditional clinical criteria clearly demonstrates the benefit of pre-treatment genotyping for all women undergoing IVF or ICSI.

[0231] Table 5. Associations of all pregnancies (fresh and frozen ET) with FSHR N680S genotype and hormones used for COH. Separate analyses of homozygous N and S allele carriers.

[0232] rFSH, (reference) vs hMG, adjusted for age and study location. CI: confidence interval. hMG: human menopausal gonadotropin. N: asparagine. OR: odds ratio. rFSH: recombinant follicle-stimulating hormone. S: serine.

[0233] Table 6. Estimated marginal mean (EMM) of aspirated oocytes in the adjusted analysis, with 95% confidence interval (CI) assessed by mean age and BMI.

[0234]

[0235] References

[0236] 1. World Health Organization. International Classification of Diseases, 11th Revision (ICD-11). Geneva: WHO; 2018. 2018

[0237] 2. Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PloS Med. 2012;9(12):e1001356

[0238] 3. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007;22(6):1506-12

[0239] 4. Rutstein SO, Shah IH. Infecundity, infertility and childlessness in developing countries. Geneva: World Health Organization; 2004

[0240] 5. ESHRE. Focus in reproduction September 2016 [Cited February 10, 2023]. Available from: https: / / www.eshre.eu / Publications / Focus-on-Reproduction / Focus-in-2016

[0241] 6. Pacchiarotti A, Sbracia M, Frega A, Selman H, Rinaldi L, Pacchiarotti A. Urinary hMG (Meropur) versus recombinant FSH plus recombinant LH (Pergoveris) in IVF: a multicenter, prospective, randomized controlled trial. Fertil Steril. November 2010;94(6):2467-9.

[0242] 7. Hugon-Rodin J, Sonigo C, Gompel A, Dodé C, Grynberg M, Binart N, et al. First mutation in the FSHR cytoplasmic tail identified in a non-pregnant woman with spontaneous ovarian hyperstimulation syndrome. BMC Med Genet. 2017;18(1):44.

[0243] 8. Uchida S, Uchida H, Maruyama T, Kajitani T, Oda H, Miyazaki K, et al. Molecular analysis of a mutated FSH receptor detected in a patient with spontaneous ovarian hyperstimulation syndrome. PloS One. 2013;8(9):e75478.

[0244] 9. Vasseur C, Rodien P, Beau I, Desroches A, Gérard C, de Poncheville L, et al. A chorionic gonadotropin-sensitive mutation in the follicle-stimulating hormone receptor as a cause of familial gestational spontaneous ovarian hyperstimulation syndrome. N Engl J Med. 2003;349(8):753-9.

[0245] 10. De Leener A, Caltabiano G, Erkan S, Idil M, Vassart G, Pardo L, et al. Identification of the first germline mutation in the extracellular domain of the follitropin receptor responsible for spontaneous ovarian hyperstimulation syndrome. Hum Mutat. 2008;29(1):91-8.

[0246] 11. Smits G, Olatunbosun O, Delbaere A, Pierson R, Vassart G, Costagliola S. Ovarian hyperstimulation syndrome due to a mutation in the follicle-stimulating hormone receptor. N Engl J Med. 2003;349(8):760-6.

[0247] 12. Kerkelä E, Skottman H, Friden B, Bjuresten K, Kere J, Hovatta O. Exclusion of coding-region mutations in luteinizing hormone and follicle-stimulating hormone receptor genes as the cause of ovarian hyperstimulation syndrome. Fertil Steril. 2007;87(3):603-6.

[0248] 13. Alviggi C, Conforti A, Santi D, Esteves SC, Andersen CY, Humaidan P, et al. Clinical relevance of genetic variants of gonadotrophins and their receptors in controlled ovarian stimulation: a systematic review and meta-analysis. Hum Reprod Update. 2018;24(5):599-614.

[0249] 14. Lledó B, Dapena P, Ortiz JA, Morales R, Llacer J, Bernabeu R. Clinical efficacy of recombinant versus highly purified follicle-stimulating hormone according to follicle-stimulating hormone receptor genotype. Pharmacogenet Genomics. 2016;26(6):288-93.

[0250] 15. Lunenfeld B, Lunenfeld E. Gonadotropic preparations–lessons learned. Fertil Steril. 1997;67(5):812-4.

[0251] 16. Wolfenson C, Groisman J, Couto AS, Hedenfalk M, Cortvrindt RG,Smitz JE, et al. Batch-to-batch consistency of human-derived gonadotrophin preparations compared with recombinant preparations. Reprod Biomed Online. 2005;10(4):442-54.

[0252] 17. Bordewijk EM, Mol F, van der Veen F, Van Wely M. Required amount of rFSH, HP-hMG and HP-FSH to reach a live birth: a systematic review and meta-analysis. Hum Reprod Open. 2019;2019(3):hoz008.

[0253] 18. Liu X, Hao C, Wang J. Efficacy of Highly Purified Urinary FSH versus Recombinant FSH in Chinese Women over 37 Years Undergoing Assisted Reproductive Techniques. Int J Fertil Steril. 2015;8(4):385-92.

[0254] 19. Kuijper EA, Blankenstein MA, Luttikhof LJ, Roek SJ, Overbeek A,Hompes PG, Twisk JW, Lambalk CB. Frequency distribution of polymorphisms in the FSH receptor gene in infertility patients of different ethnicity. Reprod Biomed Online 2010;20: 588-593.

[0255] 20. Piersma D, Verhoef-Post M, Look MP, Uitterlinden AG, Pols HA,Berns EM, Themmen AP. Polymorphic variations in exon 10 of the luteinizinghormone receptor: functional consequences and associations with breastcancer. Mol Cell Endocrinol 2007;276: 63-70.

[0256] 21. Casarini L, Moriondo V, Marino M, Adversi F, Capodanno F,Grisolia C, La Marca A, La Sala GB, Simoni M. FSHR polymorphism p.N680Smediates different responses to FSH in vitro. Mol Cell Endocrinol 2014;393:83-91.

[0257] 22. Greb RR, Grieshaber K, Gromoll J, Sonntag B, Nieschlag E, KieselL, Simoni M. A common single nucleotide polymorphism in exon 10 of the humanfollicle stimulating hormone receptor is a major determinant of length andhormonal dynamics of the menstrual cycle. J Clin Endocrinol Metab 2005;90:4866-4872.

[0258] 23. Perez Mayorga M, Gromoll J, Behre HM, Gassner C, Nieschlag E,Simoni M. Ovarian response to follicle-stimulating hormone (FSH) stimulationdepends on the FSH receptor genotype. J Clin Endocrinol Metab 2000;85: 3365-3369.

[0259] Example 3

[0260] introduction

[0261] In Examples 1 and 2, the inventors demonstrated that carriers of certain FSHR variants, when treated with hMG, had a significantly higher chance of pregnancy and live births than carriers of other variants. They were also likely to respond to hMG and produce more oocytes compared to other variants, without a higher risk of adverse outcomes. All women in the study were genotyped using standard methods: DNA extraction from blood samples, PCR amplification, and then direct sequencing. However, to improve hormone use decisions and apply new information on the importance of genotype, there is a need to develop methods for genotyping women before the initiation of hormone therapy. The inventors have developed an isothermal single-tube technique for amplifying human DNA to determine the N680S genotype of subjects. This method requires no thermal cycling or sequencing steps and can be used for on-site testing. Results can be judged visually without the need for any specialized laboratories, expensive equipment, or computer programs. Therefore, this method is ideal for clinics and healthcare providers that may not have expensive and complex equipment. Figure 2 A simplified diagram of the method is shown.

[0262] In this example, a LAMP-based methodology for FSHR N680S SNP genotyping was developed. To validate the accuracy of LAMP genotyping, the results were compared with those of Sanger sequencing.

[0263] Methods and Materials

[0264] Sample preparation

[0265] Five mL of whole blood or one mL of saliva was collected in EDTA-coated tubes and frozen (-20°C). Genomic DNA was extracted from blood samples using the Qiagen Dneasy Blood and Tissue Kit (Qiagen, Venlo, Netherlands). Saliva samples were collected using the Norgen Saliva DNA Collection, Preservation and Isolation Kit (Norgen Biotek Corp, Thorold, ON, Canada). DNA concentration and quality were determined by absorbance analysis using NanoQuant plates (Tecan, Männedorf, Schweitz).

[0266] LAMP primer design

[0267] A set of LAMP primers consists of six primers that recognize eight different regions on the target sequence. This includes one outer primer (F3, B3), one inner primer pair (FIP, BIP), and one loop primer pair (loop F, loop B). Based on the FSHR sequence, three sets of LAMP primers were designed using Primer Explorer V4 software (Primer-E Ltd., Plymouth, UK). One set was designed to be specific to the G allele and S / S genotype of the FSHR N680S SNP (FSHR N680S – G specific), and another set was designed to be specific to the A allele and N / N genotype of the FSHR N680S SNP (FSHR N680S – A specific). All inner primers had an extra mismatched nucleotide added next to the allele-specific site (both A). In addition, a set was designed to be specific to any FSHR (FSHR positive), details of which are shown in Table 7. Figure 5 A schematic diagram of the primer binding sites on the FSHR sequence is shown. All primers were synthesized by Invitrogen (Stockholm, Sweden).

[0268] Table 7. LAMP Primers

[0269] LAMP colorimetric determination

[0270] Loop-mediated isothermal amplification was performed using WarmStart colorimetric LAMP 2x premix containing UDG (New England Biolabs, MA, USA). This premix contained phenol red, a pH indicator that changed color from pink to yellow at pH < 8. The LAMP assay was prepared in a 25 μl total volume, and the components are summarized in Table 8. The premix was prepared at room temperature and incubated on a thermostat at 60–72 °C. The products were cooled to room temperature before evaluation.

[0271]

[0272] DNA sequencing

[0273] LAMP results were validated by DNA sequencing of genomic DNA from blood and saliva samples. PCR reactions were performed in a total volume of 25 μL containing 0.4 μM forward primer 5′-TCACCCCATCAACTCCTGT and 0.4 μM reverse primer 5′-TCCTGGCTCTGCCTCTTACA (Invitrogen, Stockholm, Sweden), 1.5 mM MgCl2 (Sigma AldrichSwede AB, Stockholm, Sweden), 10 mM Tris-HCl (Saveen & Werner AB) pH 9.1, 45 mM KCl (ICN Biomedicals INC.), 0.01% Tween 20 (Sharlau Chemie SA, Barcelona, ​​Spain), 200 µM of each dNTP (dATP, dCTP, dGTP, and dTTP, Fermentas, Sankt Leon-Rot, Germany), 1 U Dynazyme™ II DNA polymerase (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 200 µM of other reagents. ng template DNA. The amplification program began with a denaturation step at 96°C for 10 min, followed by 35 amplification cycles, each consisting of denaturation at 96°C for 1 min, annealing at 56°C for 30 sec, and extension at 72°C for 3 min. The program included a hotstart at 96°C and a final extension step at 72°C for 5 min. PCR products were purified using a DNA purification kit (ExtractMe DNA Purification Kit, Biolab Innovate Research Technologies, [Blirt] SA, Gda). The sample was purified (sk, Poland) and directly sequenced using the Sanger sequencing method (LightRun sequencing, Eurofins Genomics, Ebersberg, Germany).

[0274] result

[0275] LAMP was performed for 25 minutes at a temperature range of 62-68°C. 50 ng of DNA extracted from blood was used as a template. Figure 3a The results were indicated by the presence of the pH indicator phenol red. In positive reaction tubes, the color changed from pink to yellow after DNA amplification (shown in the figure as a change from dark gray to light gray). Positive signals were observed in reactions containing DNA from homozygous S / S genotypes and heterozygous N / S genotypes. Figure 3a-b). LAMP results were validated by DNA sequencing of genomic DNA from blood and saliva samples. Figure 4 This image showcases a representative image of N680S DNA Sanger sequencing.

[0276] In addition to the primers selected for subsequent use (Table 7), 11 other primers were designed and tested (Table 9).

[0277] Table 9. The LAMP primers tested did not work.

[0278]

[0279] in conclusion

[0280] We have developed a simple method to distinguish complex nucleic acid samples that differ by only a single base. This could serve as a novel diagnostic tool for detecting the asparagine-serine substitution (rs6166) in FSHR, which could be used to determine which hormone to use prior to assisted reproduction or egg donation.

[0281] SNPs constitute the main body of genetic variation among individuals, involving human diseases and normal physiological functions, such as responses to endogenous or exogenous hormones and drug responses. Due to their high similarity to non-SNP sequences, SNP detection is challenging. Direct DNA sequencing is the most reliable and commonly used method for SNP analysis. Its disadvantage is that DNA sequencing requires sample preparation and large-scale equipment, making it difficult to perform in the field or in resource-scarce environments.

[0282] For women undergoing assisted reproductive technology (ART), time is an additional limiting factor, as these patients ideally need genotyping before starting hormone therapy (which begins on day 2 of the next menstrual cycle), and traditional genotyping is time-consuming, typically taking several weeks in a routine laboratory setting. Furthermore, Sanger sequencing is expensive and therefore not routinely performed clinically. Our clinical data (Example 1) showed that using hMG in homozygous SS carriers resulted in a 15% higher pregnancy rate (hMG 41% vs. rFSH 26%, p=0.050) and a 17% higher live birth rate (hMG 33% vs. rFSH 16%, p=0.016), which is considered a significant difference in the current context. We also noted that SS women are sensitive to hMG (>15 oocytes), while homozygous NN women are sensitive to rFSH (>15 oocytes). Therefore, genotyping before ART is crucial. LAMP was originally developed for detecting viruses, bacteria, and parasites (Notomi). Waiting for someone? Nucleic Acids Res 2000;28:E63;Soroka M et al.(2021); Cells:10(8)). These strategies may be unreliable due to confounding mismatched enzymatic extension activities, leading to false positives and difficulty in distinguishing between highly similar SNPs and non-SNP target signals. Furthermore, limitations in primer / probe set design restrict the detectable SNPs (Kwong). Waiting for someone? (2018);Clinica chimica acta:;478:45-50).

[0283] Our novel LAMP method overcomes these limitations. We added an additional mutation near the SNP in the primer sequence, which greatly enhances the specificity for FSHR target SNPs. This results in a highly reliable SNP detection method applicable to both blood and saliva samples. Our LAMP-based diagnostic tool is simple, rapid, inexpensive, and can be performed on basic laboratory equipment. We believe this method is also applicable to unpurified crude nucleic acid samples. In this case, the crude sample is added to the assay reagent, providing a workflow requiring minimal manual intervention beyond a few pieces of equipment (thermostat, water bath or hot plate and thermometer).

[0284] Example 4

[0285] introduction

[0286] In Example 3, the inventors demonstrate a novel LAMP-based method for detecting the rs6166 FSHR genotype, which can be used to stratify patients for treatment with hMG or rFSH hormones prior to assisted reproduction or egg donation. This method provides an isothermal single-tube technique for amplifying human DNA to determine the subject's N680S genotype. This method requires no thermal cycling or sequencing steps and can be used for on-site testing. Results are visually interpretable and require no specialized laboratories, expensive equipment, or computer programs. Therefore, this method is ideal for clinics and healthcare providers who may not have access to expensive and complex equipment.

[0287] The inventors have demonstrated that this method is effective using oral swab samples and simplifies the DNA extraction process. Specificity and sensitivity are further significantly improved by adding a PNA clamp specific to the G or A allele of the FSHR N680S SNP to the LAMP reaction mixture. This improved method has been validated and compared with an earlier method in Example 3, as well as conventional genotyping and sequencing, as described below. Figure 6 A schematic flowchart illustrating the improved experimental method is presented.

[0288] Methods and Materials

[0289] Sample preparation

[0290] DNA was extracted from human whole blood as described in Example 2. Oral swabs were collected in Eppendorf tubes and frozen (-20°C). Before the LAMP reaction, the oral swabs were incubated in sodium hydroxide (25 mM NaOH) at 65°C for 10 minutes.

[0291] LAMP primer design

[0292] Primer sets designed to be specific to the G allele and S / S genotype (FSHR N680S-G specific) of the FSHR N680S SNP and the A allele and N / N genotype (FSHR N680S-A specific) of the FSHR N680S SNP are as described in Example 1 above (see also Table 7 and...). Figure 5 ).

[0293] PNA clamp design

[0294] To improve specificity, two peptide nucleic acid (PNA) clamps were designed and incorporated into the assay. One PNA clamp was designed to be specific to the G allele and S / S genotype of the FSHR N680S SNP, and the other was designed to be specific to the A allele and N / N genotype of the FSHR N680S SNP (Table 10). The PNA clamp function was demonstrated by... Figure 7 middle.

[0295]

[0296] LAMP colorimetric determination

[0297] Loop-mediated isothermal amplification was performed as described in Example 1. LAMP assays including PNA clamps were prepared in a 50 μL total volume containing 2 μL of DNA template (see Table 11). The mixture was prepared at room temperature and incubated on a thermostat at 65°C for 30 min. Two assays were used to validate the reaction: one using G allele-specific primers and the other using A allele-specific primers. Sensitivity for the G allele-specific assay was analyzed using DNA template concentrations ranging from 0.001 to 100 ng / μL. The G allele-specific assay was analyzed on extracted genomic DNA from 406 samples and compared with results from conventional genotyping methods (DNA sequencing). Additionally, oral swabs treated with NaOH were analyzed under the same conditions. All products were cooled to room temperature before evaluation.

[0298]

[0299] DNA sequencing

[0300] The LAMP assay results were validated by DNA sequencing of the genomic DNA from blood and saliva as described in Example 1.

[0301] result

[0302] Allele-specific LAMP

[0303] LAMP results are visualized using the pH indicator phenol red. A positive reaction is defined as DNA amplification observed in the presence of the FSHR N680S allele (specific to which this reaction assay is performed). In a positive reaction tube, the DNA color changes from pink to yellow after amplification (shown as a change from dark gray to light gray in the figure). Positive signals were observed in tubes prepared using G allele-specific primer sets in reactions containing DNA from homozygous S / S and heterozygous N / S genotypes. Positive signals were observed in tubes prepared using A allele-specific primer sets in reactions containing DNA from homozygous N / N and heterozygous N / S genotypes. LAMP results were validated by DNA sequencing of the genomic DNA. Figure 8 Results from two LAMP assays using either the G allele-specific primer set or the A allele-specific primer set are presented. The same visualizations represent results from analyzed oral swabs.

[0304] Sensitivity

[0305] The sensitivity of the LAMP assay was evaluated using G allele specificity assays with extracted FSHR N680S S / S template DNA at concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 ng / μL. The assay could detect S / S DNA down to 10 ng / μL after a 30-minute reaction. Figure 9 Visual LAMP sensitivity analysis was demonstrated.

[0306] Traditional Genotyping Comparison

[0307] 406 samples were analyzed using both G allele-specific LAMP assay and Sanger sequencing. Sequencing results showed that 120 samples were homozygous N / N, 199 were heterozygous N / S, and 87 were homozygous S / S. The G allele-specific assay correctly detected the genotypes of 386 samples, including 111 correct N / N samples and 275 correct S / S or N / S samples. Therefore, the G allele-specific LAMP assay showed a 95% concordance rate with traditional genotyping techniques.

[0308] in conclusion

[0309] The inventors have developed a simple method to distinguish complex nucleic acid samples that differ by only a single base. This can serve as a novel diagnostic tool for detecting asparagine-serine substitution (rs6166) in FSHR, which can be used to determine which hormone to use prior to assisted reproduction or egg donation.

[0310] Compared to the LAMP method described in Example 3, the addition of PNA clamps specific to the G allele and S / S genotype of the FSHR N680S SNP and the A allele and N / N genotype of the FSHR N680S SNP improved the specificity and sensitivity of this method. Furthermore, the DNA extraction procedure was simplified by replacing the time-consuming and expensive kit-based extraction method with a simple incubation at 65°C for 10 minutes under alkaline conditions (NaOH). This LAMP method also demonstrates the ability to perform patient genotyping based on oral swab samples, which are generally considered more difficult to analyze than whole blood samples.

[0311] The two LAMP-based methods described in this paper (Examples 3 and 4) overcome the limitations of traditional genotyping and sequencing methods, forming highly reliable SNP detection methods applicable to crude samples from oral swabs lysed with NaOH and DNA extracted from blood samples. Our LAMP-based diagnostic tools are simple, rapid, efficient, and inexpensive, and can be performed on basic laboratory equipment; the ability to analyze crude samples allows for minimal manual operation time, with very little equipment (thermostat, water bath or hot plate and thermometer).

Claims

1. A method for stratifying subjects to be treated with human menopausal gonadotropin (hMG) or recombinant follicle-stimulating hormone (rFSH), the method comprising or consisting of the following steps: (a) Provide samples from the subjects to be tested; (b) Determine the follicle-stimulating hormone receptor (FSHR) N680S single nucleotide polymorphism (SNP) genotype of the subject; and (c) If the genotype is homozygous or heterozygous for the 680S SNP, the subject is stratified for treatment with hMG; and if the genotype is homozygous for N680 wild-type (WT), the subject is stratified for treatment with rFSH.

2. The method of claim 1, wherein step (b) comprises detecting the presence of a polynucleotide encoding: (i) the N680S SNP of the FSHR; (ii) the T307A SNP of the FSHR; and / or (iii) the N680 WT.

3. The method of claim 2, wherein the presence of said polynucleotide is detected using a method selected from the group consisting of: polymerase chain reaction (PCR), allele-specific PCR, loop-mediated isothermal amplification (LAMP), reverse transcription LAMP (RT-LAMP), and reverse transcription PCR (RT-PCR). In situ Hybridization, nanoarrays, microarrays, sequencing, Southern blotting hybridization, or Northern blotting hybridization.

4. The method according to claims 2 to 3, wherein the detection of the presence of the polynucleotide is performed at a constant temperature.

5. The method according to any one of claims 2 to 4, wherein the presence of the polynucleotide is detected using LAMP.

6. The method according to claims 2 to 5, wherein the LAMP reaction is carried out at about 60°C to 70°C.

7. The method according to claims 2 to 6, wherein the LAMP reaction is carried out for 20 to 45 minutes, optionally for 25 to 30 minutes.

8. The method according to any one of the preceding claims, wherein the sample provided in step (a) is selected from the group consisting of saliva, oral swabs, ungraded blood, plasma, serum, tissue fluid, and urine.

9. The method according to claim 8, wherein the sample provided in step (a) is an oral swab or a saliva sample.

10. The method according to any one of the preceding claims, wherein DNA is extracted from the sample prior to step (b).

11. The method of claim 10, wherein DNA is extracted from the sample by exposure to alkaline conditions, optionally by contacting the sample with a buffer solution of pH > 8.

12. The method of claim 11, wherein the buffer solution comprises sodium hydroxide (NaOH).

13. The method according to any one of claims 10 to 12, wherein DNA is extracted from the sample by heating the sample to a temperature range of about 60°C to about 70°C.

14. The method according to any one of claims 11 to 13, wherein the exposure to alkaline conditions, the contact with the buffer solution, and / or the heating are performed for about 10 minutes.

15. A primer set for detecting 680S SNPs of FSHR, comprising polynucleotide primers containing the sequence of SEQ ID NO:2 or SEQ ID NO:

3.

16. The primer set according to claim 15, further comprising one or more of the polynucleotide primers containing the sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and / or SEQ ID NO:

7.

17. A primer set for detecting N680 WT FSHR, comprising a polynucleotide primer containing the sequence of SEQ ID NO:8 or SEQ ID NO:

9.

18. The primer set according to claim 17, further comprising one or more of the polynucleotide primers containing the sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and / or SEQ ID NO:

7.

19. The primer set according to any one of claims 15 to 18, wherein the primer set is used in the LAMP DNA amplification method.

20. The method according to any one of claims 1 to 14, wherein step (b) comprises detecting the presence of a polynucleotide encoding a 680SSNP of FSHR, and optionally a polynucleotide encoding an N680 WT FSHR. The presence of the polynucleotide was detected using LAMP. The presence of the 680S SNP of the FSHR polynucleotide was detected using the primer set described in claims 15, 16 and / or 19; optionally The presence of N680 WT FSHR was detected using the primer sets described in claims 17, 18 and / or 19.

21. The method of claim 20, wherein detecting the presence of the 680S SNP of the FSHR polynucleotide further comprises using a first nucleic acid clamp capable of annealing with the A allele of FSHR.

22. The method of claim 20 or 21, wherein detecting the presence of the N680 WT FSHR polynucleotide further comprises using a second nucleic acid clamp capable of annealing with the G allele of FSHR.

23. The method according to claim 21 or 22, wherein the first and / or second nucleic acid clamp comprises or is composed of peptide nucleic acids (PNA).

24. The method according to any one of claims 21 to 23, wherein the first nucleic acid clamp comprises or consists of the nucleic acid sequence 5'-CCAATGGTTCCACTTACA-3' (SEQ ID NO: 27).

25. The method according to any one of claims 21 to 24, wherein the second nucleic acid clamp comprises or consists of the nucleic acid sequence 5'-CCAGTGGTTCCACTTACA-3' (SEQ ID NO: 28).

26. The method according to any one of claims 1 to 14 or 20 to 25, further comprising the following steps: (d) If the subject is homozygous for the 680S SNP or heterozygous for the FSHR, then administer hMG to the subject; and / or (e) If the subject is homozygous N680 WT FSHR, then rFSH is administered to the subject.

27. The method according to any one of claims 1 to 14 or 20 to 26, wherein the subject is female.

28. A method for treating involuntary childlessness in a subject, the method comprising the steps of: (a) The method according to any one of claims 1 to 14 and 20 to 25, wherein the subjects are stratified for treatment with hMG or rFSH; (b) administering hMG or rFSH to the subjects according to the stratification; and (c) Obtaining oocytes from the subject for assisted reproduction.

29. A method for reducing the risk of ovarian hyperstimulation syndrome (OHSS) in subjects receiving ovarian stimulation, the method comprising the steps of: (a) The method according to any one of claims 1 to 14 and 20 to 25, wherein the subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

30. The method according to any one of claims 27 to 28, wherein the number of oocytes obtained from the subject and / or the achieved increase in pregnancy.

31. The method according to any one of claims 1 to 14 and 20 to 26, wherein the subject is male.

32. A method for treating infertility in male subjects, the method comprising the following steps: (a) The method according to any one of claims 1 to 14 and 20 to 25, wherein the subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

33. The method according to any one of claims 31 to 32, wherein the subject has increased testosterone levels, sperm count, sperm concentration and / or a lower DNA fragmentation index after hMG or rFSH treatment.

34. The primer set according to any one of claims 15 to 19, used for treating infertility or reducing the risk of OHSS in subjects receiving ovarian stimulation, said use comprising the following steps: (a) The method according to any one of claims 20 to 25, wherein the subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

35. Human menopausal gonadotropin (hMG) or recombinant follicle-stimulating hormone (rFSH), used in subjects undergoing controlled ovarian hyperstimulation to treat infertility or reduce the risk of OHSS, said use comprising the following steps: (a) The method according to any one of claims 1 to 14 and 20 to 25, wherein the subjects are stratified for treatment with hMG or rFSH; (b) Administer hMG or rFSH to the subject according to the stratification.

36. The primer set according to any one of claims 15 to 19 for use in stratifying subjects for treatment with hMG or rFSH using the method according to any one of claims 1 to 14 and 20 to 25.

37. The method or use according to any one of claims 1 to 14 and 20 to 36, wherein the method or use is in in vitro conduct.

38. The method or use according to any one of claims 1 to 14 and 19 to 37, wherein the subject is experiencing involuntary childlessness, optionally wherein the subject suffers from infertility.

39. A kit for determining the FSHR 680S SNP genotype of a subject, the kit comprising: (i) The primer set according to any one of claims 15 to 19; (ii) DNA polymerase, optionally included in the master mixture; (iii) Phenol red; and (iv) (Optional) DNA lysis buffer.

40. The kit according to claim 39, further comprising: (iv) swab (v) A description of the method for performing any one of claims 1 to 14 and 20 to 25.

41. Basically as described in this document: methods, uses, or kits.

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