A method for the determination of the likelihood of getting pregnant based on measuring the stnfr1 level

Abstract

The invention discloses a method for the determination of the likelihood of getting pregnant based on measuring the sTNFR1 level in a sample collected from a female patient tested.

Description

[0001] A method for the determination of the likelihood of getting pregnant based on measuring the sTNFRI level

[0002] The invention discloses a method for the determination of the likelihood of getting pregnant based on measuring the sTNFRI level in a sample collected from a female patient tested.

[0003] T umor necrosis factor receptor 1 , also known as tumor necrosis factor receptor superfamily member 1A and CD120a, is a ubiquitous membrane receptor that binds tumor necrosis factor alpha (TNF-a). When cleaved by TACE proteinase (TNF-a converting enzyme), membrane TNFa as well as its receptors (TNFR1 and TNFR2) occur in the soluble form (sTNFR, Soluble Tumor Necrosis Factor Receptor) in a number of body fluids. Elevated sTNFR levels have been found in serum, plasma, and urine due to endotoxemia, infections, and malignancies as well as chronic autoimmune diseases. TNF receptor shedding may result in transiently reduced cellular response to TNF-a. It has been suggested depending on sTNFR levels that sTNFRs block TNF-a binding with transmembrane TNFR, and thus delay biological responses. However, it may also be that TNFR shedding in inflammatory conditions may initiate direct signaling effects through sTNFR binding to transmembrane TNF-a (mTNF-a) [Idriss HT, Naismith JH. TNF alpha and the TNF receptor superfamily: structure-function relationship(s). Microsc Res Tech. 2000 Aug 1 ;50(3): 184-95; Georg H Waetzig et al. Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-betal . FASEB J. 2005 Jan; 19(1 ):91 -3].

[0004] The role of sTNFRI has been reported in pre-eclampsia, i.e. pregnancy-induced hypertension, in several studies [Nevalainen J et al. Performance of first-trimester biochemical markers and mean arterial pressure in the prediction of early-onset pre-eclampsia. Metabolism. 2017 Oct;75:6-15; Leal AM et al. First-trimester maternal serum tumor necrosis factor receptor-1 and pre-eclampsia. Ultrasound Obstet Gynecol 2009;33:135-41 ; Estensen ME et al. Elevated inflammatory markers in preeclamptic pregnancies, but no relation to systemic arterial stiffness. Pregnancy Hypertens. 2015 Oct; 5(4):325-9; Yliniemi A et al. Combination of PAPPA, fhCGP, AFP, PIGF, sTNFRI , and Maternal Characteristics in Prediction of Early-onset Preeclampsia. Clin Med Insights Reprod Health. 2015 Jun 11 ;9:13- 20; Minuz P et al. Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia. J Matern Fetal Neonatal Med. 2015 May; 28(8):869-75; Laskowska M et al. Maternal and umbilical sTNF-R1 in preeclamptic pregnancies with intrauterine normal and growth retarded fetus. Hypertens Pregnancy. 2007; 26(1 ):13-21], The role of sTNFRI has also been reported in early miscarriage [Yu XW et al. Tumour necrosis factor-alpha receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage. Cell Biol Int. 2007 Nov; 31 (11 ): 1396-9; Chernyshov VP et al. Lack of soluble TNF- receptors in women with recurrent spontaneous abortion and possibility for its correction. Am J Reprod Immunol. 2005 Nov; 54(5):284-91].

[0005] Infertility is currently a growing problem seen globally and in Poland. It is estimated to affect between 8 and 12% of pairs at childbearing age worldwide [Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21 st century. Hum Reprod Update. (2015) 21 :411-26]. Fertility rates have been decreasing globally since 1960. The decreased fertility results from the fact that women now later decide to get pregnant and postpone maternity. The average age of a woman who had her first baby was 29.7 years in Poland in 2015 (Statistics Poland, 2015). Unfortunately, with increasing age women face decreasing likelihood of natural conception and maintaining pregnancy. Many women require assisted reproduction techniques (ART), the last chance for pairs with infertility to have a baby. However, despite the advancement of intracytoplasmic sperm injection (ICSI), the rate of successful embryo implantation is surprisingly low. The success rate of IVF in 2005 and 2008 was 25 and 28%, respectively. The rate increased to 32% in 2016, which did not indicate significant progress in this area. Unfortunately, recurrent implantation failure (RIF), affecting 10-15% of pairs who had several in vitro fertilizationembryo transfers (IVF-ET), is the most frustrating problem in terms of economic and psychological aspects for embryologists and infertile pairs. The most widely accepted definition of RIF is no pregnancy after at least 3 transfers of good-quality embryos [Busnelli A, et al. How common is real repeated implantation failure? An indirect estimate of the prevalence. Reprod Biomed Online. 2019 Oct 29. Pii:S1472-6483(19)30790-4], The etiology of RIF may be attributed to the embryo itself, the mother or the father, and both in some cases. Other important factors include oocyte and sperm quality, chromosomal anomalies affecting the parents, stage of embryo development and number of embryos transferred [Coughlan C, et al. Recurrent implantation failure: definition and management. Reprod Biomed Online. (2014) 28:14-38]. The maternal factors affecting RIF include congenital uterine abnormalities, intrauterine adhesions and endometrial polyps, and endometriosis. In addition, immune factors, such as levels of pro- and anti-inflammatory cytokines, presence of specific autoantibodies as well as any uterine infections, may result in chronic endometritis. Therefore, some clinicians have administered steroids to suppress inflammation. However, long-term steroid therapy may cause side effects in the female body as well as result in congenital defects of the embryo and the fetus [Busada JT, Cidlowski JA. Mechanisms of Glucocorticoid Action During Development. Curr Top Dev Biol. 2017;125:147-170; Painter RC, Roseboom TJ, de Rooij SR. Long-term effects of prenatal stress and glucocorticoid exposure. Birth Defects Res C Embryo Today. 2012 Dec;96(4):315-24],

[0006] The objective of the invention was to develop a method for increasing the likelihood of getting pregnant and maintaining pregnancy in patients who had in vitro fertilization, in particular those experiencing multiple failures of embryo implantation and using steroid therapy.

[0007] The object of the invention is a method for the determination of the likelihood of getting pregnant, characterized in that the sTNFRI level is measured in a blood sample collected from the patient tested, and the likelihood of getting pregnant is considered high:

[0008] - after measuring an sTNFRI level greater than 1409 sTNFRI pg / mL in the plasma from a female who has not taken steroid therapy or

[0009] - after measuring an sTNFRI level greater than 962.3 sTNFRI pg / mL in the plasma from a female who has taken steroid therapy.

[0010] The female tested preferably had an in vitro fertilization procedure.

[0011] In the method of the invention, the female tested preferably had luteal support by the intravaginal administration of 2 x 200 mg micronized progesterone and oral administration of 3 x 10 mg dydrogesterone for a period until 12 weeks of pregnancy.

[0012] In the method of the invention, the female tested preferably had treatment including the administration of 5 mg prednisone once daily, preferably in the morning by mouth, starting on the day of embryo transfer (ET) into the uterus.

[0013] In the method of the invention, the patient with recurrent embryo implantation failure preferably received a steroid in increased doses (10-20 mg) for 2-3 weeks before embryo transfer and for up to 8 weeks when pregnancy developed after the transfer.

[0014] A different object of the invention is the use of the sTNFRI level measurement, in particular in blood, to determine the likelihood of getting pregnant.

[0015] The object of the invention is illustrated by the following embodiments and a drawing, wherein:

[0016] Figure 1 shows the concentration of soluble TNFR1 per mL of plasma (pg / mL) measured before and after embryo transfer in all IVF patients and the fertile control group

[0017] (A) Concentration of soluble TNFR1 (pg / mL) measured before and after embryo transfer in patients treated and not treated with steroids and in fertile control (without steroid therapy); (B) Concentration of soluble TNFR1 (pg / mL) measured before and after embryo transfer in patients treated with steroids who became clinically pregnant, without pregnancy, with miscarriage, and fertile control females (without steroid therapy). sTNFRI : soluble TNFR1. Red dots indicate measurement before embryo transfer; green dots: after embryo transfer; dark gray: fertile control with a history of giving birth; light gray: fertile control during pregnancy. N is the number of females. Black horizontal lines are medians. P values were calculated using the Mann-Whitney test.

[0018] Figure 2 shows the analysis of ROC curves

[0019] (A) Comparison of patients who got pregnant after in vitro fertilization and maintained pregnancy with those who failed to get pregnant or had miscarriage. AUC (area under curve), red dot: borderline value.

[0020] (B) Comparison of patients who did not get pregnant after in vitro fertilization with fertile females with a history of giving birth. AUC (area under curve), red dot: borderline value.

[0021] Role of sTNFRI in embryo implantation failure

[0022] Research has shown that the higher concentration of circulating sTNFR, the lower bioavailability of TNF-a, which may reduce inflammatory response. It was unexpectedly found that this was related to whether steroid administration to patients with higher sTNFRI levels was reasonable. sTNFRI secretion to plasma was found before getting pregnant and at a very early stage of pregnancy (2-3 weeks after embryo transfer to the uterus) in patients who had an in vitro fertilization procedure. The women were treated with steroids and progesterone.

[0023] 1. Preparing patients to the IVF procedure, luteal support and steroid therapy

[0024] Patients undergoing a standard IVF procedure were instructed to routinely take 5 mg prednisone (Encorton, Adamed, Poland) once a day in the morning by mouth, starting on the day of embryo transfer (ET) to the uterus. Patients with recurrent embryo implantation failure (RIF) received the steroid in increased doses (10-20 mg) for 2-3 weeks before ET and for up to 8 weeks when pregnancy developed after the transfer. To supplement the luteal phase, the patients were requested to take 2 x 200 mg micronized progesterone (Lutein, Adamed, Poland) intravaginally and dydrogesterone 3 x 10 mg by mouth (Duphaston, Solvay Pharmaceuticals, The Netherlands) until 12 weeks of pregnancy. The patients had 3 embryo transfer failures on average and were eligible for inclusion in the RIF group. 2. Measuring plasma sTNFRI concentrations

[0025] Plasma samples were collected from patients after in vitro fertilization (IVF) before (before ET) and after embryo transfer (after ET) into the uterus. The second plasma collection occurred when the human chorionic gonadotropin beta subunit (P-hCG) was determined to confirm pregnancy. Plasma was also collected from fertile women who had a history of giving birth to a healthy child after natural conception and from women who were pregnant at the time of specimen collection for testing. The samples were stored at -80°C until tested. sTNFRI concentration (pg / mL) was measured using a commercial Human ProcartaPlex kit (Thermo Fisher Scientific, USA) using Luminex 200 (Luminex Corp., USA).

[0026] Determination of pro- and anti-inflammatory cytokines and factors in plasma

[0027] Cytokines and pro- and anti-inflammatory factors in plasma may be determined using any method. In the aforementioned embodiment, the ProcartaPlex immunoassay (Thermo Fisher Scientific) was used, based on the Luminex xMAP technology which involves simultaneous detection and quantitative determination of one to several tens of target proteins in a single sample of plasma, serum, cell culture supernatants and other body fluids. The Luminex technology is based on dyed magnetic microbeads coated with antibodies that capture specific analytes via ELISA. Each ProcartaPlex kit contains standards of known concentration to determine the standard curve.

[0028] Human ProcartaPlex assay

[0029] With the commercial Human ProcartaPlex (Thermo Fisher Scientific) kit, the following 7 analytes could be simultaneously measured in plasma: BDNF, IL-6, PIGF, LIF, TNF-RI, VEGF-A, VEGF-D. The measurements were performed according to the assay manufacturer’s instructions.

[0030] Concentrations of the cytokines and factors were measured in plasma from females who had IVF (before and after embryo transfer) and from fertile control females. The test started with the preparation of standards in serial dilutions according to the manufacturer’s protocol and the thawing of plasma samples. A solution with magnetic beads was added to a 96-well plate. The plate was placed on a manual magnetic separator and the magnetic beads were rinsed with the Wash Buffer after 2 min. The rinsing involved adding 150 pL of the Wash Buffer into a plate in the separator and subsequently removing the buffer and the plate was taken out from the magnetic separator only after the removal. Standards (50 pL) and plasma (25 pL) with the universal buffer (25 pL) were applied on the plate. The plate was incubated at room temperature for 2 hours on a shaker at 500 rpm. Thereafter, the plates were rinsed twice. 25 pL of detection solution (Detection Antibody) was added and incubated for 30 min on a shaker (500 rpm). Rinsing twice was repeated. Streptavidin (50 pL) was added and the incubation was performed again (30 min, 500 rpm). Subsequently, the plate was rinsed twice and 120 pL of the Reading Buffer was added and briefly incubated (5 min, 500 rpm). The results were read using Luminex 200.

[0031] 3. sTNFRI secretion in patients after in vitro fertilization and the fertile group

[0032] 149 patients and 19 patients who took steroids and did not take steroids, respectively, were tested. There were no significant differences in sTNFRI secretion between the steroid-treated and untreated patients (Fig. 1A, Table 1 ). The greatest amount of sTNFRI was secreted by fertile women with a history of giving birth (median 1843 pg / mL). They differed in their plasma sTNFRI concentrations from the IVF patients treated with steroids (p = 0.0069, median 1843 vs. 962.80 pg / mL before embryo transfer), and those taking steroids (p < 0.0001 , median 1843 vs. 676.50 pg / mL before embryo transfer; p = 0.0007, median 1843 vs. 998.70 pg / mL after transfer).

[0033] The lowest sTNFRI concentration among the patients taking steroids was found in patients who did not get pregnant (median 0.00 and 143.30 pg / mL, measurements before and after ET ; Fig. 1 B, Table 2). The patients who got pregnant secreted higher levels of sTNFRI than those who did not (p<0.0001 , median 1134 vs. 0.00 pg / mL, measurement before ET; p = 0.0015, median 1207.00 vs. 143.30 pg / mL, measurement after ET) and those who had miscarriage (p = 0.0066, median 1134.00 vs. 414.70 pg / mL, measurement before ET and p = 0.0504, median 1207.00 vs. 721.30 pg / mL, measurement after ET). The group of females who had miscarriages differed from those who did not get pregnant, either (p = 0.042, median 414.70 vs. 0.00 pg / mL, measurement before ET). The most significant differences were found when comparing patients with no pregnancy and fertile females with a history of giving birth (p < 0.0001 , median 0.00 before ET vs. 1843 pg / mL and p < 0.0001 , median 143.30 after ET vs. 1843 pg / mL). In addition, the patients secreted less sTNFRI than the control group of fertile pregnant females (p=0.0005, median 0.00 pg / mL before ET vs. 523.80 pg / mL and p=0.0162, median 143.30 pg / mL after ET vs. 523.80 pg / mL).

[0034] 4. ROC analysis in terms of sTNFRI secretion

[0035] In addition, the ROC (receiver operating characteristics) curve was analyzed for sTNFRI secretion. A sTNFRI threshold value of 962.3 pg / mL was determined to distinguish IVF patients who got pregnant and maintained pregnancy from those who did not get pregnant or had a miscarriage (AUC=0.66, p=0.00186, sensitivity 62.71 %, specificity 62.16% and likelihood ratio, LR=1 .66; Fig. 2A). When comparing fertile females with a history of giving birth and those who did not get pregnant or had miscarriage, the threshold value was 1409 pg / mL, AUC = 0.76, p < 0.0001 , sensitivity 77.97%, specificity 69.23%, LR = 2.53; Fig. 2B). Conclusions: Females who secrete at least 1409 pg / mL sTNFRI have a chance of getting pregnant naturally and giving birth to a child, while those after in vitro fertilization with simultaneous luteal support and steroid therapy need to achieve a plasma sTNFRI concentration of 962.3 pg / mL.

[0036] Table 1 Concentration of soluble TNFR1 (pg / mL) measured before and after embryo transfer in patients after in vitro fertilization (IVF) treated and not treated with steroids and in the control group

[0037] ET : embryo transfer; p values calculated using the Mann- Whitney test

[0038] Patients not treated with steroids before ET vs. fertile control:ap = 0.0069

[0039] Patients treated with steroids before ET vs. fertile control:bp < 0.0001

[0040] Patients treated with steroids after ET vs. fertile control:cp = 0.0007

[0041] Test group IVF patients Fertile Fertile

[0042] „ „ . . . . control control

[0043] Treatment No steroids Steroids . . during before or after IVF-ET before after before after pregnancy

[0044] Number of women 19 13 149 133 39 27

[0045] Minimum 0.00 0.00 0.00 0.00 0.00 0.00

[0046] 25% percentile 425.50 1033.00 0.00 140.80 910.60 311.70

[0047] Median 962.80a1177.00 676.50b998.70c1843.00 523.80

[0048] 75% percentile 1389.00 1536.00 1685.00 1687.00 2444.00 2100.00

[0049] Maximum 2279.00 2785.00 3948.00 4123.00 4956.00 4575.00

[0050] Mean 962.50 1291.00 926.80 1066.00 1850.00 1280.00

[0051] Standard deviation 620.70 645.00 972.40 985.60 1320.00 1419.00

[0052] Standard error 142.40 178.90 79.67 85.46 211.40 273.10

[0053] Lower 95% CI for mean 663.30 901.60 769.40 896.80 1422.00 718.50

[0054] Higher 95% CI for mean 1262.00 1681.00 1084.00 1235.00 2278.00 1841.00

[0055] ^D'Agostino & PearsonQ 45 4 w 16 34 n 97 2 59 6 59normality test

[0056] Table 2 Concentration of soluble TNFR1 (pg / mL) measured before and after embryo transfer in patients after in vitro fertilization (IVF) treated with steroids who got pregnant or did not get pregnant and had miscarriage and in the control group

[0057] ET : embryo transfer; p values calculated using the Mann- Whitney test:

[0058] Pregnancy before ET vs. no pregnancy before ET:ap < 0.0001

[0059] Pregnancy before ET vs. miscarriage before ET:bp = 0.0066

[0060] Pregnancy before ET vs. fertile control:cp = 0.0341

[0061] Pregnancy after ET vs. no pregnancy after ET: “ p = 0.0015

[0062] Pregnancy after ET vs. miscarriage after ET:ep = 0.0504

[0063] Pregnancy after ET vs. fertile control:fp = 0.0395

[0064] No pregnancy before ET vs. miscarriage before ET:gp = 0.0420

[0065] No pregnancy before ET vs. fertile control:11p < 0.0001

[0066] No pregnancy before ET vs. fertile control during pregnancy:1p = 0.0005

[0067] No pregnancy after ET vs. fertile control:Jp < 0.0001

[0068] No pregnancy after ET vs. fertile control during pregnancy:kp = 0.0162

[0069] Miscarriage before ET vs. fertile control:1p < 0.0001

[0070] Miscarriage after ET vs. fertile control:mp = 0.001

[0071] Test group IVF patients treated with steroids Fertile Fertile

[0072] Result Pregnancy No pregnancy Miscarriage control control before after before after before afterpregnancy

[0073] Number of women 75 74 37 25 37 34 39 27

[0074] Minimum 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

[0075] 25% percentile 211.50 431.20 0.00 0.00 76.99 94.97 910.60 311.70

[0076] Median 1134.00a l,’c1207.00“’e’f(LOO8’11’' 143.30fk414.70' 721.30m1843.00 523.80

[0077] 75% percentile 2017.00 2047.00 600.80 1207.00 1409.00 1511.00 2444.00 2100.00

[0078] Maximum 3948.00 4123.00 3265.00 2785.00 2368.00 2252.00 4956.00 4575.00

[0079] Mean 1283.00 1320.00 447.40 584.50 684.00 866.20 1850.00 1280.00

[0080] Standard deviation 1052.00 1054.00 756.70 796.00 693.40 778.00 1320.00 1419.00

[0081] Standard error 121.40 122.50 124.40 159.20 114.00 133.40 211.40 273.10

[0082] Lower 95% CI for1041 001076.00 195.10 256.00 452.80 594.80 1422.00 718.50 mean

[0083] Higher 95% CI for1525 00 1564.00 699.70 913.10 915.20 1138.00 2278.00 1841.00 mean

[0084] K2D'Agostino & Pearson normality 4.46 4.52 27.25 8.54 5.57 7.72 2.59 6.59 test

Claims

Claims1. A method for the determination of the likelihood of getting pregnant, characterized in that the sTNFRI level is measured in a blood sample collected from the patient tested, and the likelihood of getting pregnant is considered high:- after measuring an sTNFRI level greater than 1409 sTNFRI pg / mL in the plasma from a female who has not taken steroid therapy or- after measuring an sTNFRI level greater than 962.3 sTNFRI pg / mL in the plasma from a female who has taken steroid therapy.

2. The process of claim 1 , characterized in that the female tested had an in vitro fertilization procedure. The process of claim 1 , characterized in that the female tested had luteal support by the intravaginal administration of 2 x 200 mg micronized progesterone and oral administration of 3 x 10 mg dydrogesterone for a period until 12 weeks of pregnancy.

4. The process of claim 1 , characterized in that the female tested had treatment including the administration of 5 mg prednisone once daily, preferably in the morning by mouth, starting on the day of embryo transfer (ET) into the uterus.

5. The process of claim 1 , characterized in that the female tested with recurrent embryo implantation failure received a steroid in increased doses (10-20 mg) for 2-3 weeks before embryo transfer and for up to 8 weeks when pregnancy developed after the transfer.

6. The use of the sTNFRI level measurement, in particular in blood, to determine the likelihood of getting pregnant.

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