AUTOLOGOUS SOMATIC STEM CELL THERAPY, METHOD OF CONTROLLABLE PREPARATION OF THERAPEUTIC COMPOSITION AND PROCEDURE OF ADAPTIVE TREATMENT OF IVF PATIENT

Abstract

A blood product containing peripheral blood mononuclear cells (PBMCs) in an amount of at least 4 million cells per milliliter and human chorionic gonadotropin (HCG in an amount of at least 150 international units (IU) per milliliter. A method of preparing the blood product, including applying HCG to a female patient, then obtaining PBMCs from the female patient, then adding HCG to the obtained PBMCs. A method of culturing PBMCs, including applying HCG to a female patient, then culturing PBMCs obtained from the female patient at a time after the HCG was applied to the patient. A method of in vitro fertilization, including applying HCG to a female patient, culturing PBMCs obtained from the patient after the HCG was applied to the patient, introducing the cultured PBMCs into the uterus of the patient, and transferring at least one embryo into the uterus of the patient.

Claims

1-19 (canceled)

20. A composition produced by a method comprising: isolating a first portion of isolated peripheral blood mononuclear cells (PBMCs) from the blood of a female patient, and culturing the PBMCs in vitro in the presence of at least 500 IU/ml of HCG; further comprising administering HCG at a time T0 to the female patient in an amount sufficient to maintain a serum HCG level of 150-350 IU/ml until the first portion of PBMCs is isolated at a time T1; wherein said composition comprises the first portion of PBMCs at a concentration of at least 15×10.sup.6 cells per milliliter that have been obtained from a female patient at time T1, and human chorionic gonadotropin (HCG) at a concentration of at least 500 international units (IU) per milliliter.

21. The composition of claim 20, wherein the female patient is intramuscularly administered HCG at time T0 in an amount sufficient to maintain serum HCG at 150-350 IU/ml until time T1 and wherein the time T1 is 30 to 40 hours after the time T0.

22. The composition of claim 20, further comprising culturing the first portion of isolated PBMCs in vitro in a medium containing a concentration of at least 500 international units/ml of HCG from a time Ti until a time T2, wherein T2-T1 ranges from 20 to 80 hours.

23. The composition of claim 20, further comprising measuring a concentration of HCG in the PBMC culture at least once during the period from T1 to T2, and, optionally introducing at least one additional amount of HCG into the culture during this period.

24. The composition of claim 20, further comprising a second fresh portion of PBMCs isolated from the female patient at a time T2 after the collection of the first portion of PBMCs at time T1, wherein T2-T1 ranges from 20 to 80 hours, wherein the first and second portions are combined in a medium containing at least 500 IU/ml of HCG; and resuspended at a concentration of at least 15×10.sup.6PBMCs/ml.

25. The composition of claim 20, wherein, prior to culturing, the first portion of PBMCs is processed to approach a larger proportion of CD14.sup.+ monocytes or CD14.sup.+-HCG linked monocytes compared to the unprocessed.

26. The composition of claim 20, wherein, after culturing, said composition is further processed to approach a larger proportion of CD14.sup.+ monocytes or CD14.sup.+-HCG linked monocytes compared to the unprocessed.

27. A method of in vitro fertilization comprising administering the composition of claim 20 to the uterus of a female patient prior to transferring an embryo into the uterus.

28. The method of claim 27, further comprising intramuscularly injecting HCG into the patient.

29. The method of claim 27, wherein the female patient is suffering from recurrent implantation failure, autoimmune infertility, idiopathic infertility, or is greater than 37 years of age.

30. A composition comprising: a first portion of isolated peripheral blood mononuclear cells (PBMCs) at a concentration of at least 15×10.sup.6 cells per milliliter that have been obtained from a female patient at a time T1, and human chorionic gonadotropin (HCG) at a concentration of at least 500 international units (IU) per milliliter.

31. The composition of claim 30, further comprising selecting the first portion of PBMCs to comprise CD14.sup.+ monocytes or CD14.sup.+-HCG linked monocytes in a larger proportion compared to a proportion of CD14.sup.+ monocytes in PBMCs from blood of the female patient not exposed to exogenous HCG.

32. The composition of claim 30, wherein the female patient has been administered HCG in vivo at a time T0 prior to isolation of the first portion of PBMCs at the time T1.

33. The composition of claim 30, wherein the female patient has been administered HCG in an amount sufficient to maintain a serum HCG level of 150-350 IU/ml in vivo at the time T1.

34. The composition of claim 30, wherein the female patient has been administered at least one of CRH, GnRH-A, or HCG so as to maintain a serum HCG level in a range 150-350 UI/ml

35. The composition of claim 30, wherein the first portion PBMCs have been cultured in vitro in a medium containing a concentration of at least 500 international units/ml of HCG from a time T1 until a time T2, wherein T2-T1 ranges from 20 to 80 hours, wherein said medium optionally contains IL, LIF or TKA.

36. The composition of claim 30, wherein the female patient has been administered HCG at a time T0 prior to the time T1, wherein T0 to T1 ranges from about 30 to 40 hrs.

37. The composition of claim 30, further comprising a second portion of PBMCs obtained from the female patient at the time T2, wherein said second portion of PBMCs has been mixed with the first portion of PBMCs in a medium containing a concentration of at least 500 international units/ml of HCG.

38. The composition of claim 30, wherein the female patient is additionally administered HCG after time T1 and wherein a second portion of PBMCs is collected after time T1.

39. A method of in vitro fertilization comprising administering the composition of claim 30 to the uterus of a female patient prior to transferring an embryo into the uterus and, optionally, further comprising intramuscularly injection HCG into the patient.

40. The method of claim 39, wherein the female patient is suffering from recurrent implantation failure, autoimmune infertility, idiopathic infertility, or is greater than 37 years of age.

41. The method of in vitro fertilization of claim 39, wherein said composition is administered into the uterine cavity of the female patient via a catheter.

42. The method of in vitro fertilization of claim 39, wherein said catheter is suitable for intrauterine injection and for embryo transfer.

43. The method of in vitro fertilization of claim 39, wherein said composition is delivered into the uterine cavity via said catheter in a dose ranging from 0.1 to 0.35 mL.

44. The method of in vitro fertilization of claim 39, wherein said composition is delivered into the uterine cavity via said catheter in a dose that has a volume ranging from 0.1 to 0.35 mL and said dose contains at least 165 IU of HCG.

45. The method of in vitro fertilization of claim 39, wherein said composition is delivered into the uterine cavity via said catheter in a dose that has its volume ranging from 0.1 to 0.35 mL and said dose contains at least 2.25 million PBMCs.

46. The composition of claim 20, wherein said composition is in a form of a suspension having a viscosity allowing it to flow through a catheter medically-acceptable for an intrauterine injection to a female patient.

47. A catheter that is preloaded with the 0.1 to 0.35 mL of the composition of claim 20 that is calibrated for delivery into the uterine cavity.

48. The catheter of claim 47 wherein said composition comprises or further comprises a dose of HCG of 165 IU or more.

49. The composition of claim 30, wherein said composition is in a form of a suspension having a viscosity allowing it to flow through a catheter medically-acceptable for an intrauterine injection to a female patient.

50. A catheter that is preloaded with the 0.1 to 0.35 mL of the composition of claim 30 that is calibrated for delivery into the uterine cavity.

51. The catheter of claim 50, wherein said composition comprises or further comprises a dose of HCG of 165 IU or more.

52. A kit comprising at least one catheter for intrauterine transfer of an embryo to a patient, a glassware, HCG, and, optionally, other material components for performing IVF.

53. A kit for preventing or reducing the likelihood of implantation failure in a recipient of in vitro fertilization, the kit comprising a first device for preparation a portion of HCG, a second device for a culture media, a third device for detection and measurement, a fourth device for injection, and optionally label marks, a portable computer, and a sensor for reading radio frequency identification (RFID).

54. The kit of claim 53, wherein the third device is for measurement of HCG and comprises a glassware, reagents and other material components necessary for a quantitative detection and measurement of HCG, wherein the glassware comprises pipettes, flasks, tubes and optionally other laboratory glassware, wherein the detection is intended to be performed by a 2-methoxyethanol (CLIA) method, enzyme-linked immunosorbent assay (ELISA) method, Förster resonance energy transfer (FRET) method, immuno-magnetic reduction (IMR) assay method or other method of quantitative determination of a concentration of HCG in plasma, serum, urine, cervicovaginal secretion, amniotic fluid of a patient, an embryo culture medium or PBMC culture medium.

55. The kit of claim 53, wherein the third device is for detection and measurement of a PBMC concentration and comprises glassware, reagents and other material components necessary for counting of a number of PBMCs per a volume, wherein the glassware comprises pipettes, flasks, tubes and optionally other laboratory glassware, wherein the counting is intended to be performed manually by a hemocytometry counter or automatically by a impedance counter or a flow cytometry counter or by other method for determination of a PBMC number per volume of a liquid specimen or a suspension specimen.

56. The kit of claim 53, wherein the fourth device for injection is a catheter for intrauterine transfer of an embryo to a patient.

57. The kit of claim 53, wherein the fourth device for injection is a syringe for intramuscular injection of HCG to a patient.

58. The kit of claim 56 further comprising an additional catheter identical to the catheter for intrauterine transfer of an embryo, wherein the additional catheter is capable of intrauterine injection of a composition comprising PBMCs and HCG into a patient's uterine cavity.

59. The kit of claim 57 further comprising an additional syringe identical to the syringe for intramuscular injection of HCG, wherein the additional syringe is configured for adding HCG into the culture medium or for taking a sample of the culture medium for measurement.

60. A method of preventing or reducing the likelihood of implantation failure in a female patient undergoing in vitro fertilization (IVF), comprising: (a) administering a dose of HCG to said patient prior to embryo transplantation, (b) preparing an initial culture medium (ICM) that contains HCG, (c) separating a first portion of peripheral blood mononuclear cells (PBMCs) from the patient's blood, (d) culturing said first portion of PBMCs in the ICM for a time and under conditions that produce an active culture medium (ACM) containing the cultured PBMCs and HCG, (f) separating a second fresh portion of PBMCs from a separate sample of the patient's blood, (g) putting said second fresh portion of PBMCs into said ACM thereby obtaining a composition comprising both cultured and fresh PBMCs and HCG, and producing a dose of said composition, (h) analyzing HCG, PBMCs or other detectable biomarker in said patient's blood, culture medium, composition or other IVF-related biospecimens and, based on said analyzing, making a decision regarding a complementary action most suitable for preventing or reducing the likelihood of implantation failure.

61. The method of claim 60, wherein the analyzing is performed prior to or in course of any of (a) to (g) and comprises measurements of HCG, PBMCs or said biomarker, wherein the decision depends on said measurements' results be in ranges of desirable values of HCG, PBMC and said biomarker, wherein said desirable values are known a priori, and wherein the complementary action comprises at least one of: (i) making no changes in (a) to (g), perform an intrauterine injection of the dose of the composition to the patient and perform the embryo transfer after said injection; (ii) making at least one change in (a) to (g), perform an intrauterine injection of the dose of the composition to the patient and perform the embryo transfer after said injection; (iii) considering (a) to (g) as a training session for better preparing of the female patient to the implantation, postpone the embryo's thawing and reschedule the embryo transfer to another cycle of ovulation of the patient.

62. The method of claim 60, wherein two or more measurements of HCG are performed during the culturing.

63. The method of claim 60, wherein the analyzing comprises measurement of at least one isoform of HCG.

64. The method of claim 60, wherein the analyzing comprises measurement of monocyte CD14, other specific sort of PBMC or a combination of thereof, and optionally comprises measurement of CD14.sup.+ with linked HCG.

65. The method of claim 61, wherein pattern of the desirable values for the patient is determined by statistical calculation based both on known results of one or more training sessions performed with the patient and known results of previously accomplished attempts of IVF that were applied to plurality of other IVF patients, wherein said pattern allows to predict most suitable applying of the changes, and wherein this makes higher a probability of successful embryo implantation.

66. The method of claim 61, wherein the changes comprise increasing or decreasing the dose of HCG in (a), increasing or decreasing content of HCG in the ICM of (b), increasing or decreasing the culture time in (e), adding cells of a donor's blood in (e) or (f), applying additional input of HCG in (e) or (g), and applying other means that allow to nearing of the measurements' results of HCG, PBMC and other biomarkers to their desirable values known a priori.

67. The method of claim 60, further composing conducting the complementary action, thereby preventing or reducing implantation failure.

Description

EXAMPLES

[0211] Infertile couples were admitted to clinical treatment. Among those admitted, the male factor of infertility was excluded. Some female patients had a history of RIF containing at least three unsuccessful IVF attempts including fresh- and cryo-IVF.

[0212] Oocytes were retrieved from the female patients using standard procedures known to those of ordinary skill in the art. Embryos were created using a variant of intracytoplasmic sperm injection (ICSI), and developmental kinetics were measured. Embryos were cryopreserved by vitrification.

[0213] Major steps, ingredients, conditions and duration of stages of the process of culturing, as well as procedures of administering the blood product are described in U.S. patent application Ser. No. 13/655,257, incorporated herein by reference.

[0214] As in U.S. patent application Ser. No. 13/655,257, the culture in the present examples contained 5 mL of commercially available media RPMI1640, 0.2 mL of commercially available human recombinant albumin, 1 mL of autologous PBMC and 1 mL of solution having 5000 IU of diluted HCG (obtained by dilution of commercially available dry powder of HCG), such that the total volume of culture was equal to 7.2 mL.

[0215] Differences between the procedures used in the present examples and those of U.S. patent application Ser. No. 13/655,257 were as follows.

[0216] In the examples in the present application, PBMCs were obtained from the blood of the patients (as in U.S. patent application Ser. No. 13/655,257). However, in the present examples, HCG was applied to the patients prior to obtaining the blood from which the PBMCs were obtained. In other words, the patients were pre-activated with HCG, prior to blood being taken from the patients for collection of PBMCs.

[0217] Additionally, in the examples in the present application, measurements were taken of the concentration of HCG, and, depending on the results of the measurements, changes in the procedure could be implemented in real time. In the present examples, for implementing changes in the culture, a device with a prepared solution of HCG was kept ready on standby.

Example 1 (Comparative)

[0218] A total of 180 female patients were treated, 90 according to the traditional ART protocol and 90 according to a modified protocol with the use of PBMC+HCG (as described in U.S. patent application Ser. No. 13/655,257; i.e., without intentionally targeted pre-activating the patients with HCG). All patients were of age 32 to 42 years, and had experienced three or more unsuccessful IVF attempts in the past. Two high quality embryos were transferred to every patient at every IVF attempt, fresh or cryo.

[0219] The results are summarized in the following Table 1, reproduced from U.S. patent application Ser. No. 13/655,257:

TABLE-US-00001 TABLE 1 Group 1 (without PBMCs) Group 2 (with PBMCs) Fresh- 22.2% 31.1% IVF (20 pregnancies after 90 ET) (28 pregnancies after 90 ET) Cryo- 21.4% 41.9% IVF (15 pregnancies after 70 ET) (26 pregnancies after 62 ET) Total 38.9% 60.0% (35 pregnancies for 90 patients) (54 pregnancies for 90 patients)

[0220] As seen in Table 1, a combined PBMC+HCG therapy showed success in overcoming RIF, especially for patients undergoing cryo-IVF.

Example 2

[0221] Building on the success of Example 1, the inventors undertook a more comprehensive evaluation of the influence of HCG-dose, moment of time of the dose's administration, and other process parameters on the implantation success rate.

[0222] Eleven patients obtained a course of PBMC+HCG treatment similar to Example 1, prior to fresh embryo transfer (ET). Unlike in Example 1, however, the patients were intentionally pre-activated with HCG prior to blood being taken from the patients for collection of PBMCs. Four of the 11 patients had one or more poor quality embryos at the time of transferring, and no pregnancy was achieved among these four patients. For the remaining seven patients, embryos of high quality were transferred. Results for these seven patients are in the following Table 2:

TABLE-US-00002 TABLE 2 8 9 10 11 1 2 3 4 Absolute Relative ? Dose of * Dose of Pat. Age Att. En 5 6 7 increase increase beta-hCG total hCG 12 No. years No. mm C.sub.0 C.sub.24 C.sub.48 C.sub.48 − C.sub.0 C.sub.48/C.sub.0 0.2 × C.sub.48 IU Result 1 33 2 11 4.8 5.2 9.6 4.8 2.0 1.92 300 Pregnancy 2 34 1 9 4.2 5.5 8.8 4.6 2.1 1.76 275 Negative 6 40 1 9 0.9 — 6.3 5.4 7.0 1.26 200 Negative 7 45 4 8 4.1 5.5 7.9 3.8 1.9 1.58 250 Negative 8 39 1 10 3.1 — 7.4 4.3 2.4 1.48 230 Pregnancy 9 34 5 9 5.9 — 10.1 4.2 1.7 2.02 315 Pregnancy 11 27 1 10 0.8 — 5.2 4.4 6.5 1.04 160 Bio-Chemic

[0223] Table 2 includes measurements of the conditional concentration of beta-hCG in culture media, in IU/ml. In this context, “conditional” means that the measurement in culture media was performed by the test recommended for measuring beta-HCG in serum. Measurements were performed by beta-HCG ELISA assay (DRG Instruments GmbH, Germany). Numeration of patients (column 1) is in chronological order (patient No. 1 is the earliest one). In column 2 , the patient's age is shown; in column 3 , the number of IVF attempts; and in column 4, the thickness of endometrium prior to ET.

[0224] Measurements of the conditional concentration of beta-hCG were taken at different time points: C.sub.0 was the initial concentration prior to the input of PBMC; C.sub.24 was the concentration after 24 hours of culturing; and C.sub.48 was the concentration after 48 hours of culturing, prior to adding a fresh portion of PBMC. The volume of each culture was: 5 ml RPMI media+0.2 ml albumin+1 ml HCG solution+1 ml PBMC=7.2 ml.

[0225] As seen in Table 2, the success rate (resulting pregnancies treated patients) was 37 or 43%.

[0226] The average value of initial conditional beta-concentration (column 5, excluding patients 6 and 11) was C.sub.0=4.42 IU/ml.

[0227] The average absolute increase of a conditional beta-concentration, in IU/ml, was 4.3 (column 8 of Table 2). The average relative increase (column 9, excluding patients 6 and 11) was 2.2 times.

[0228] Column 11 shows an estimate of the total dose of HCG delivered prior to ET. The estimated dose is calculated in accordance with the formula: Dose=V×K.sub.beta×C.sub.48, where V is a volume of delivered PBMC mixture equal to 0.2 ml, and K.sub.beta is an empirical coefficient establishing a correspondence between the measured conditional beta-HCG and the real total HCG.

[0229] K.sub.beta can be determined from the relationship between the average value of C.sub.0 and total HCG as follows: K.sub.beta×C.sub.0=5 000 IU/7.2 ml. Since the average value of C.sub.0 was found to be 4.42 IU/ml, K.sub.beta is 157. This value was used to estimate the total dose of HCG delivered prior to ET shown in column 11 of Table 2.

[0230] Of the seven patients in Table 2, successful implantation was achieved in three cases (1, 8, 9). In the other cases, (2, 6, 7, 11) the implantation was unsuccessful, including an unfortunate patient number 11, for whom a biochemical test showed a positive indication two weeks after ET, but three weeks after ET, the ultrasound test had not confirmed initiation of pregnancy.

[0231] Quantitative examination of data from Table 2 shows a correlation between HCG-parameters and the success rate of implantation.

[0232] Indicator: Cutoff value of concentration of beta-HCG at moment of time T0: C.sub.0=4.8 IU/ml. Two groups: C.sub.0.sup.+≥4.8 for (1, 9), rate=2/2=100%; C.sub.0.sup.−<4.8 for (2, 6, 7, 8, 11), rate=15=20%.

[0233] Indicator: Cutoff value of concentration of beta-HCG at moment of time T2: C.sub.48=9.6 IU/ml. Two groups: C.sub.48.sup.+≥9.6 for (1, 9), rate=22=100%; C.sub.48.sup.−<9.6 for (2, 6, 7, 8, 11), rate=15=20%.

[0234] Indicator: Cutoff value of dose of total HCG injected to the patient: Dose=300 IU. The same two groups as if considering the indicator C.sub.48, because Dose=V×K beta×C.sub.48.

Example 3

[0235] Twenty-four patients obtained a course of PBMC+HCG treatment prior to fresh embryo transfer, similar to Example 2. Three of the 24 patients had poor quality embryos at the time for transferring, therefore it was decided to interrupt the procedure without performing ET on these three patients. For one other patient (out of the 24), a genetic analysis of an embryo suggested certain complications, and ET was not performed. For the remaining twenty patients, embryos of high quality were transferred. Results for these twenty patients are shown in Table 3 below:

TABLE-US-00003 TABLE 3 Pat. Age, Att. Endome- Day 0 Day 1 Day 2.1 Day 2.2 Dose No. years No. trium, mm C.sub.0 C.sub.24 C.sub.47 C.sub.49 IU Result 2 28 1 10 8.67 11.10 7.64 8.74 93 N 3 26 1 9 8.95 6.72 6.59 12.50  132 N 5 38 2 10 9.73 12.67 10.82 11.10  118 pregnancy 6 41 4 9 8.98 10.67 13.64 14.39  152 N 7 31 1 10 11.77  15.47 13.04 14.35  152 N 8 41 1 10 12.43> 14.01 14.00 16.67> 177 N 9 35 1 8 12.76> 13.77 11.64 12.07  128 pregnancy 10 40 1 8 14.69> 9.68 15.83 19.93> 211 pregnancy 11 41 5 11 13.34> 14.09 15.27 11.36  120 pregnancy 12 38 1 9 11.20  12.24 14.35 16.33> 173 pregnancy 13 43 3 10 13.85> 12.86 12.05 15.02  159 N 14 32 2 9 9.81 11.92 10.11 15.21  161 N 15 34 1 9 12.41> 11.27 11.94 15.74> 167 pregnancy 16 34 3 9 15.66  14.99 17.03 — ? pregnancy 17 40 1 9 19.87> — 15.51 13.79  146 N 18 35 1 8 13.32  18.52 19.59 — ? N 19 36 1 10 14.80> — 14.99 17.38> 184 N 21 28 3 11 17.98> — 17.36 20.75> 220 pregnancy 23 46 1 7 17.36  15.31 17.81 — ? N 24 29 4 13 14.33> — 16.33 12.65  134 N

[0236] Table 3 includes measurements of the conditional concentration of total HCG in culture media, in IU/ml. In this context, “conditional” means that the measurement in culture media was performed by the test recommended for measuring total HCG in serum. Measurements were performed by HCG ELISA assay (DRG Instruments GmbH, Germany). Numeration of patients (column 1) is in chronological order (patient No.1 is the earliest one). In column 2 , the patient's age is shown; in column 3 , the number of IVF attempts (including six RIF patients); and in column 4, the thickness of endometrium prior to ET.

[0237] Measurements of the conditional concentration of total HCG were taken at different time points: C.sub.0 was the initial concentration prior to the input of PBMC; C.sub.24 was the concentration after 24 hours of culturing; C.sub.47 was the concentration after 48 hours of culturing, prior to adding a fresh portion of PBMC, and C.sub.49 was the concentration after 48 hours of culturing, immediately after adding a fresh portion of PBMC. The volume of each culture was: 5 ml RPMI media+0.2 ml albumin+1 ml HCG solution+1 ml PBMC=7.2 ml. The total dose was calculated in a manner similar to Example 2, but using the average initial value C.sub.0=13.1 from Table 3. This resulted in an estimate for K of 53 from the relationship of K×C.sub.0=5 000 IU 7.2 ml. The total dose then was calculated as 0.2×K×C.sub.49.

[0238] As seen in Table 3, the success rate (resulting pregnancies treated patients) was 8/20 or 40%.

[0239] Considering those patients who had two or more unsuccessful attempts prior to PBMC+HCG treatment, there were 6 RIF patients, and 3 became pregnant, for a rate of 3/6 or 50%.

[0240] Considering those patients for whom the present treatment was the second attempt of IVF, there were 2 patients, and 1 became pregnant, for a rate of 1/2 or 50%. This was the same as for the RIF patients above.

[0241] Considering those patients for whom the present treatment was the first attempt of IVF, there were 12 patients, and 4 became pregnant, for a rate of 4/12 or 33%.

[0242] Thus, the rate difference between first time treated IVF patients and others was 50/33=1.5 times. This shows that there is a certain difference in the rate of first-time treated patients and RIF patients.

[0243] Quantitative examination of data from Table 3 shows a correlation between HCG-parameters and the success rate of implantation. The patients (16, 18, 23) were excluded from further investigation, because there was no C.sub.49 measurement accomplished.

[0244] Among 17 patients with all measurements, seven were positive. Rate=7/17=41%.

[0245] Indicator: C.sub.0=12.4 IU/ml (C.sub.0.sup.+ corresponds to C.sub.0≥12.4 and C.sub.0.sup.− corresponds to C.sub.0<12.4). C.sub.0.sup.+=(8, 9, 10, 11, 13, 15, 17, 19, 21, 24)=>5/10=50%; C.sub.0.sup.−=(2, 3, 5, 6, 7, 12, 14)=>2/7=29%.

[0246] Rate difference with respect to this indicator=50/29=1.7 times

[0247] The following recommendation comes from these data: If C.sub.0<12.4 then add some HCG to approach a positive indicator on Day 0.

[0248] Indicator: C.sub.49=15.5 IU/ml (C.sub.49.sup.+corresponds to C.sub.49≥15.5 and C.sub.49.sup.− corresponds to C.sub.49<15.5). C.sub.49.sup.+=(8, 10, 12, 15, 19, 21)=>4/6=67%; C.sub.49.sup.−=(2, 3, 5, 6, 7, 9, 11, 13, 14, 17, 24)=>3/11=27%;

[0249] Rate difference with respect to this indicator=67/27=2.5 times

[0250] The following recommendation comes from these data: If C.sub.47 or C.sub.49<15.5, then add HCG or wait up to 24 hours additionally before performing ET.

[0251] Six patients had both indicators negative: C.sub.0.sup.− and C.sub.49.sup.−=(2, 3, 5, 6, 7, 14). Only one of these six (patient number 5) became pregnant. Rate=16=17%. Conclusion: Two simultaneously negative indicators predict an implantation failure with probability of 83%.

[0252] Five patients had both indicators positive: C.sub.0.sup.− and C.sub.49.sup.+=(8, 10, 15, 19, 21). Three of these five became pregnant. Rate=35=60%=> Approaching of such dynamics is desired. For patients 8 and 19, who had both indicators positive but who did not get pregnant, it appeared that an autoimmune situation might be operative, and further cryogenic protocol might be appropriate.

Example 4

[0253] Patient Number 9 of Table 2.

[0254] Patient 9 was a white married woman of age 34. Prior to admission to the PBMC+HCG treatment, she had four unsuccessful attempts of assisted reproductive technique (ART), one of which was fresh embryo transfer (ET) and three others were ET after cryo-preservation of embryos. None of these four attempts indicated even a bio-chemical pregnancy.

[0255] The fifth ART attempt was initiated with use of PBMC+HCG administration in March 2013. Both indicators C.sub.0 and C.sub.48 were achieved positively. Three embryos of high quality were transferred, the biochemical test showed a positive result two weeks after ET, and the ultrasound test confirmed a definitive pregnancy for one embryo three weeks after ET.

[0256] As of August 2013, a pregnancy of Patient 9 was ongoing under observation of fertility professionals.

Example 5

[0257] Patient Number 21 of Table 3)

[0258] Patient 21 was a white married woman. She had been a patient of the inventors' fertility clinic since 2011. As of 2011, there had been no pregnancy after over 2 years of marriage. Her husband's spermatogenesis, quantity and motility of spermatozoa, as well as genetic diagnostics, were without abnormalities. The factor of male infertility was almost excluded.

[0259] In 2011 the assisted reproduction technique in a form of artificial intrauterine insemination was applied, but did not lead to pregnancy.

[0260] In the beginning of 2012, an IVF cycle with “fresh” transfer of two embryos was performed; the thickness of endometrium was 13 mm as measured prior the transferring. One of these two embryos had linked to the endometrium, and clinical pregnancy was confirmed by the ultrasound test three weeks after ET. However, at the very early stage of this pregnancy, a spontaneous abortion happened (a few days after the ultrasound test).

[0261] In the second half of 2012, another IVF attempt with transfer of two thawed embryos after cryopreservation was performed; the thickness of endometrium was 12 mm then. This time there was no implantation success demonstrated (either by biochemical test or by ultrasound).

[0262] After the unsuccessful attempts above, the technology of PBMC+HCG was proposed to Patient 21 in 2013, and she was admitted to participate in a new IVF cycle. The recent Fresh-IVF procedure was performed in April 2013; Patient 21 was 28 years old then. After preliminary administration of PBMC+HCG, three high quality embryos were introduced into the uterine cavity having its endometrium of 11 mm. All three were successfully implanted as confirmed by ultrasound.

[0263] At the 7th week, the reduction of one embryo was executed. As of August 2013, the 17th week of pregnancy was proceeding with two remaining embryos and Patient 21 in good condition.

[0264] In all IVF attempts, only the embryos of high quality were transferred, and the thickness of endometrium was in the range from 11 to 13 mm. Thus, in case of Patient 21, not only the quality of embryo and thickness of endometrium were important, but also the mediating influence of the PBMC+HCG composition on the interaction of maternal body with semiallograft fetus was so helpful that resulted in a successful implantation and definitive pregnancy.

[0265] As it follows from the tables and examples above, the PBMC+HCG treatment according to embodiments of the invention delivers a similar or better success rate for RIF patients, compared with first-time IVF patients. This demonstrates the ability of the invention to overcome certain problems of RIF.

[0266] Without being bound by theory, the clinical results might be explained as follows. After culturing in HCG-enriched media, the PBMCs of a non-pregnant woman can themselves begin production of HCG and, therefore, the intrauterine delivery of such PBMCs prior to ET is capable of improving the endometrium preparation for ET, improving the maternal body's immunotolerance to ET, and overall increasing the probability of successful implantation of the embryo.

[0267] Differences between the doses that are shown in Tables 2 and 3 may be explained as follows. Estimations were performed with the use of empirical coefficients K.sub.beta (Table 2) and K (Table 3). The time of measurement (prior to or after adding fresh PBMC), the design of tests that are appropriate for measurements of HCG in serum, and the interaction of some components of the media with the test's reagents are possible reasons of observations reflecting such differences.

[0268] Additionally, there is a certain diversity in ELISA assays produced by dozen of manufacturers; some of assays can measure just one specific isoform of HCG; others can measure several isoforms; and some interfering is possible. The precision of measurements may be non-uniform for different isoforms measured by different assays. A discussion about metrological standard has been continuing for a long time. [Sturgeon et al 2009; Whittington et al & Grenache 2010; Cole 2013].

[0269] Despite these difficulties with empirical coefficients and diversity in ELISA assays, the inventors have demonstrated that a time-controlled treatment based on observed HCG dynamics both in culture media and in a patient's blood samples is effective for IVF.

Example 6

[0270] The above examples illustrate the improved success rate in treating IVF patients using embodiments of the present invention. In particular, the above examples show that RIF patients can benefit from such treatment. Although RIF can occur in younger patients, it tends to correlate with a patient's age. That is, among older females (age 38 years and over) who apply for IVF, a substantial portion of them suffer from RIF.

[0271] Based on the demonstrated ability of PBMCs to treat RIF, and the known fact that many older patients suffer from RIF, the inventors hypothesized that the age-related risk of implantation failure may overlap, in etiology, with the risk of RIF. Accordingly, in the following example, the inventors analyzed the usefulness of PBMC treatment for all patients over 38 years of age, independently of the number of previous IVF attempts.

[0272] In a clinical trial, a PBMC treatment was offered (at a subsidized price) to all IVF female patients of age ≥38 to ≤45 years with BMI of 18 to 32 kgm2 and a normal uterine cavity without uterine disease (visible on pelvic ultrasonography), who had at least two top quality embryos (own, non-donated) for the transfer. Exclusion criteria were: any serious systemic disease or endocrine disorders (e.g., diabetes mellitus or untreated thyroid dysfunction); uterine myoma or previous myomectomy; presence of hydrosalpinges; also endometriosis in the severe form.

[0273] Patients who agreed to take the PBMC treatment formed a treatment group (N=94) and proceeded to the study under IRB approval; those who declined were the control (N=39). The subgroups were: 49 patients of age 38-40 in PBMC group vs 24 in control; 26 patients of age 41-42 in PBMC group vs 12 in control; also 19 patients of age 43-45 in PBMC group vs 3 in control; as shown in Table 4 below.

[0274] Embryo transfer (ET) of two top quality embryos was performed 24 to 72 hours following PBMC injection. After such ET, clinical pregnancy was determined by means of standard ultrasound diagnostics. Chi-squared test was used for statistical analysis of differences between the patient groups.

[0275] Prior to taking the patient's blood samples for separation of PBMC, an intramuscular injection of HCG was administered in a certain amount per dose.

[0276] A first amount of HCG was administered prior to obtaining a first portion of fresh blood from a patient, and a second amount of HCG was administered prior to obtaining a second portion of fresh blood from the same patient. The first portion of the patient's blood was used to prepare a first blood product according to an embodiment of the invention, while both the first and second portions of the patient's blood were used to produce a second blood product according to an embodiment of the invention.

[0277] Second blood products of autologous PBMC with human chorionic gonadotropin (HCG) were produced and applied prior to embryo transfer (ET) in fresh- and cryo-IVF protocols similar to those described in U.S. application Ser. No. 13/655,257. However, the present example did not categorize fresh vs cryo cases. The composition was delivered through a catheter as an intrauterine injection in an amount of 0.15 mL of a suspension containing PBMC cells and HCG. The results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Age Age Age Preg- Pregnancy Group 38-40 41-42 43-45 Total nant Rate PBMC 49 26 19 94 25 25/94 = 26.6% (Preg) (30.6%) (23.1%) (21.1%) Control 24 12  3 39 8  8/39 = 20.5% (Preg) (25.0%) (16.7%) (0.0%)

[0278] As seen in Table 4, the clinical pregnancy rate in the PBMC group (26.6%) was 30% higher than in the control group (20.5%). 2594 patients became pregnant in the PBMC group vs 839 patients in the control (p=0.46).

[0279] These observations were rather favorable for the use of PBMC, especially since the oldest subgroup of 43-45 years of age was disproportionally larger in the PBMC group, than in the control group (1994 vs 339). Among the oldest patients, there were 419 pregnancies with PBMC use and 03 without it (p=0.38).

[0280] The pregnancy rate (PR) decreased with age in both the PBMC and control groups of patients. However, among the PBMC-treated patients, the PR decreased less sharply compared to the control group.

[0281] These results demonstrate a tendency of steady increasing of the gap between the PR of PBMC-treated patients and the PR of control patients with increasing patient age. These PR were 30.6% vs 25.0% for ages 38-40, 23.1% vs 16.7% for ages 41-42 and 21.3% vs 0% for the oldest subgroup of ages 43-45 in the trial. Thus, when stratified by age, subgroups of older patients had a larger gap between the success rate in treated and control patients compared to the gap for younger patients.

[0282] These results suggest a benefit of intrauterine use of PBMC+HCG for females over 38, and this is a promising improvement compared to traditional IVF. Without being bound by theory, the inventors hypothesize that the reproductive system of older female patients requires a longer time to prepare for acceptance of the arriving embryo in IVF treatment. The use of PBMC+HCG prior to ET leads to 1) early signaling of immune cells to the mother's body to be prepared on the systemic level, and 2) early preparation of endometrium environment on the local level. These two mechanisms may provide effective overcoming of age-related unpreparedness for ET and, therefore, result in better inducing of pregnancy in case of PBMC-enhanced IVF treatment, compared to conventional one.

[0283] It is natural to expect that an older organism works slower in immuneendocrine aspects. However, if a clinician initiates preparation for embryo acceptance in advance of its arriving, even a slow-working organism can be ready at the proper time. Again without being bound by theory, the injection of PBMC+HCG might mimic the presence of an embryo inside the patient even before the embryo is present. In this way, the preparation for embryo acceptance begins in advance; hence, when the embryo actually arrives, the organism is already tuned to attach it to the endometrium. Such an understanding is built on the facts that: 1) HCG is the first cytokine that embryo is producing for earliest signaling to mother's organism about its presence; 2) HCG is an active participant of fetal-maternal cross talk important to implantation; 3) embryo presence induces the endometrium for local secretion of HCG in return; 4) HCG mediates a cascade of the organism's processes that are important to pregnancy inception, maintaining and development; particularly, HCG modulates several endometrial parameters as endometrial differentiation (IGFBP-1), angiogenesis (VEGF), implantation (LIF, M-CSF) and tissue remodeling (MMP-9); 5) HCG can be bound with certain cells of PBMC variety, be released by such cells, and be transported by means of PBMC throughout the organism; 6) rapid increase of HCG concentration in female's fluids is an important attribute of healthy pregnancy inception; and finally 7) bio-chemical pregnancy test is based on the measurement of HCG, exactly.

[0284] This example shows that the age-related risk of implantation failure can be significantly reduced by using embodiments of the present invention, compared to using traditional IVF.