Use of soluble CD146 as a biomarker to select in vitro-fertilized embryo for implantation in a mammal

Abstract

The present invention relates to the field of human fertility treatment. The present invention more specifically relates to the identification of soluble CD146 (sCD146) as a biomarker which, when measured in an embryo culture medium, can be used to determine whether the embryo can be selected for implantation in the uterus of a mammal or not. The present invention thus provides a new tool and related kits to (pre)select embryo eligible for implantation. The invention also relates to methods for promoting pregnancy in a human who undergoes embryo transfer.

Claims

1. A method for promoting pregnancy in a mammal undergoing embryo transfer comprising culturing embryos in a culture medium, collecting the culture medium after at least one day of embryo culture, measuring the quantity of soluble CD146 (sCD146) protein in the collected culture medium, and transferring to said mammal one or more embryos collected from culture medium in which the measured quantity of sCD146 is equal to or below a threshold value.

2. The method of claim 1, said method comprising a step of examining the cultured embryos using morphological criteria and transferring at least one embryo that satisfies morphological criteria of implantation into the uterus of said mammal.

3. The method of claim 1, wherein the quantity of soluble CD146 is measured between two and five days after in vitro oocyte fertilization.

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Distribution of couples and embryos.

(2) FIG. 2: Western blot analysis of positive or negative embryos supernatants by ELISA test.

(3) Negative control corresponds to culture medium without any embryo. MW, molecular weight.

(4) FIG. 3: sCD146 concentrations at day 2 (D2) and day 3 (D3).

(5) FIG. 4: sCD146 concentrations according to the embryo quality as defined by the classification of Istanbul (type 1 top n=90, type 2 fair n=190, or type 3 poor n=43).

(6) FIG. 5: Comparison of sCD146 concentrations between implanted (YES, n=63) and non-implanted embryos (NO, n=172).

(7) FIG. 6: Percentage of pregnancy according to the value of sCD146.

EXAMPLES

(8) Material and Methods

(9) Patients

(10) From March 2013 to December 2014, inventors performed an initial pilot study on 162 couples who underwent In Vitro Fertilization (IVF) attempts in the reproductive department of medical center at La Conception University Hospital (AP-HM, Marseille, France). All couples were informed that embryo culture media from transferred embryos would be preserved after embryo transfer for research purposes, and chose to participate or not to this study. Each couple was included once only. Inventors excluded from this study oocyte and sperm donors and patients with lack of consent. The Institutional Review Board approved this investigation.

(11) Treatment Protocol

(12) Patients underwent a controlled ovarian hyperstimulation using three types of protocols: long agonist protocol (GnRH agonist administration in the luteal phase of the previous cycle); short agonist protocol (daily GnRH agonist administration since the first day of the IVF cycle); and antagonist protocol (daily GnRH antagonist administration from Day 5). Recombinant FSH and/or hMG were used at doses ranging between 150 IU/day and 450 IU/day, in accordance with body mass index, woman's age, basal day 3 FSH value and number of antral follicles. Patients routinely underwent serial transvaginal ultrasound starting on Day 8 of ovarian hyperstimulation and serum estradiol (E2) measurements. The dose of gonadotropin was then adjusted according to the ovarian response. Ovulation triggering was performed with subcutaneous injection of recombinant human Chorionic Gonadotrophin (hCG, Ovitrelle, Merck-Serono, 250 g) when at least three follicles reached a mean diameter of 16 mm. Oocyte retrieval was carried out under local or general anesthesia using transvaginal ultrasound-guided puncture of follicles 35 hours after hCG administration. Conventional IVF or IntraCytoplasmic Sperm Injection (ICSI-IVF) was then carried out according to sperm parameters, using routine protocols. After assessment of normal fertilization (i.e. fertilized oocytes with two pronuclei, so-called diploid zygotes) 18 h post-insemination, zygotes were then individually cultured at 37 C., 5% CO2 in 500 l of Global medium (Life Global) until the day of embryo transfer (Day 2 or 3).

(13) They were observed 26 hours post-insemination in order to detect the early cell-cleavage. Obtained diploid embryos were graded before transfer according to scoring system based on cell size and symmetry, fragmentation and cell number according to Consensus of Istanbul (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. Hum Reprod. 2011).

(14) Embryos were then classified in 3 subgroups:

(15) 1/ Top quality embryos with equally sized cells, no fragmentation or less than 10%, and 4 cells on day 2 or 8 cells on day 3; preferentially selected for transfer when available;

(16) 2/ fair quality embryos with stage-specific cell size for majority of cells and/or 10-25% fragmentation, and/or 3 or 5 cells on Day 2 or 6, 7 or 9 cells on day 3;

(17) 3/ Poor quality embryo with no stage specific cell size and/or severe fragmentation (>25%) and/or 2 cells or >5 cells on Day 2 or <6 or >9 cells on day 3.

(18) After embryo transfer, luteal phase was then supported by daily progesterone tablets (DuphastonR 30 mg/day; Abbot Products SAS, France). Pregnancies were diagnosed by serum positive hCG levels (>100 IU/l) 14 days after embryo transfer. Clinical pregnancies were confirmed by the presence of a gestational sac with cardiac activity on vaginal ultrasound examination during the 5th week after embryo transfer.

(19) Embryo Supernatants

(20) Culture media (500 l Global) of each transferred embryo were collected individually after embryo transfer, frozen and stored at 20 C. until sCD146 quantification. Collecting embryos supernatants is a non-invasive technique; there was no change in the care of patients. IVF outcomes were retrospectively compared in all patients.

(21) Confirmation of the Presence of sCD146 in Embryo Supernatants

(22) CD146 has already been identified in early stages of human embryo (Wang H et al. J of Reprod and contracept 2008) and it is known that a soluble form can be generated by shedding of membrane CD146 in trophoblastic cells (Kaspi et al., Angiogenesis, 2013). However, no data is available on secretion of sCD146 by embryos in the literature. Inventors carried out western blot analysis on embryo supernatant to confirm the release of sCD146 by embryos. 50 L of embryo supernatants or negative control (culture medium) were submitted to 4-12% NuPage SDS-polyacrylamide gel electrophoresis (In Vitrogen/Life Technologies, USA) and transferred onto nitrocellulose membrane. Bio-Rad molecular weight markers were used. Transfer was performed at constant voltage (60 V) for 2 hours. After blocking with 4% Bovine Serum Albumine (BSA) in TBS-Tween 20 (TBST), soluble CD146 was evidenced with anti-human CD146 antibody (7A4 1 mg/l, Biocytex, Marseille, France) diluted in TBST (1:3000), overnight at 4 C. with constant shaking. Soluble CD146 was revealed by HRP-coupled goat anti-mouse antibody (Thermo Scientific, USA). Membranes were scanned and analyzed by the G:Box-Chemi-XT4 (Syngene, Cambridge, United Kingdom).

(23) Soluble CD146 Assay

(24) sCD146 was assayed using an adaptation of the commercial ELISA assay (CY-QUANT sCD146, Biocytex, Marseille). Plates were coated with specific mouse monoclonal anti-human CD146 F(ab)2 fragments. 200 L of embryo supernatant diluted was added to each well and incubated for 30 minutes at room temperature. After incubation, the plates were washed five times, followed by incubation with a specific HRP-coupled anti-CD146 monoclonal antibody (7A4-HRP, 1 mg/ml, Biocytex, Marseille, France) at a 1:1 000 dilution in a specific diluent for 30 minutes at room temperature and then washed five times. 200 of tetramethylbenzidine (TMB) substrate was incubated for approximately 20 minutes at room temperature. The colorimetric reaction was then stopped by the addition of 100 L of an acid solution. The intensity of the signal was directly related to the concentration of sCD146 initially contained in the sample. Adaptation of the technique was based on the substitution of the diluent of the kit by embryo culture medium, conserved in the same conditions as embryo supernatants (37 C., 5% CO2 for 48 h), but without embryo in order to improve repeatability and reproducibility of the test. A concentrated anti-CD146 antibody was also used because of lower concentration of sCD146 in supernatants than in human serum or plasma (7A4-HRP, 1 mg/ml, Biocytex, Marseille, France). Concentrations of sCD146 in the embryo supernatants were determined using a calibration curve of solutions with known concentrations of sCD146 (from 0 pg/ml to 10 000 pg/ml). Due to the low volume of supernatants and the supplier's recommendations (200 l/well), each sample was diluted (1:2) and analyzed in simplicate. Optical density (OD) was measured at 450 nm.

(25) A repeatability and reproducibility analysis was performed and data showed 4% of repeatability and 11% of reproducibility (n=3 tests).

(26) Collected Data

(27) Inventors collected patient's clinical and biological data (age, body mass index, smoking habit, indication and duration of infertility, assessment of ovarian reserve evaluated by antral follicles count and basal Day 3 FSH and AMH plasma levels), characteristics of the IVF cycle and laboratory data (number of previous IVF-ICSI attempts, conventional IVF or ICSI-IVF, ovarian stimulation protocol, estradiol levels and endometrial thickness on the triggering day, number of retrieved oocytes, of mature oocytes, of diploid embryos obtained and of embryo transferred, morphologic score of embryos transferred, the day of transfer), and occurrence of clinical pregnancies.

(28) Statistical Analysis

(29) Data were expressed as meanSEM. Statistical analysis was performed with the Prism software (GraphPad Software Inc., San Diego). Significant differences were determined using non parametric Mann Whitney and Chi2 tests. Because we have several observations for each couple, a generalized estimating equation (GEE) with a multivariate model is used. A p-value<0.05 was considered significant.

(30) Results

(31) Baseline Patient Characteristics

(32) The study group included 162 couples who underwent IVF or IVF-ICSI with at least one transferred embryo. Of the 1 505 retrieved oocytes, 1 199 were mature (81.2%), 907 embryos were obtained and 738 were diploid. On day 2/3, a total of 261 embryos were transferred, with one or two embryos per transfer. Out of the 162 couples studied, complete data (sCD146 test result and pregnancy testing) were available for 138 of them with 225 transferred embryos (FIG. 1). These 225 transferred embryos resulted in 36 clinical pregnancies and included 33 singletons and 3 twins. 146 embryos were transferred at day 2 (D2, 64.8%), 79 at day 3 (D3, 35.2%). The mean number of embryos transferred was 1.63 (SD+/0.53) and the implantation rate was 17.3% (39 sacs/225 embryos). The overall pregnancy rate was 26.1% (36 pregnancies/138 patients).

(33) Among these 225 embryos, and according to Istanbul classification, 64 were top quality embryos, 125 fair quality and 36 poor quality. Their implantation rate was of 26.2%, 12% and 5.7% respectively.

(34) Women's mean age was 33.1 years (SD+/4.51). Controlled ovarian hyperstimulation was performed using short agonist protocol in 21.5% of attempts, long agonist protocol in 58.3% and antagonist protocol in 20.2%. 54% of the couples underwent classical in vitro fertilization, 46% intracytoplasmic sperm injection. The mean row of IVF cycles was 1.77 (SD+/0.97). Indications for IVF were related to male infertility for 58% of couples or to female infertility for 42%. Characteristics of the studied population (ovarian reserve status, clinical prognosis factors of implantation, ovarian response and endometrial status on the triggering day) are summarized in Table 1.

(35) TABLE-US-00001 TABLE 1 Characteristics of the studied population. Data are expressed as mean +/ SD and median [IQR] AMH (ng/mL) 3.68 +/ 3.38 2.50 [1.36-5.20] FSH (UI/L) 7.452 +/ 2.601 7 [5.6-8.78] Antral follicles count 14.32 +/ 8.74 12 [9-18] Body mass index (BMI, Kg/m.sup.2) 23.81 +/ 4.98 22.5 [20.7-26.0] Current smokers (%) 28 History of previous pregnancy (%) 35 Estradiol (pg/mL) on the triggering day 2394 +/ 1392 2216 [1495-2946] Endometrial thickness on the triggering day Good quality: 8-13 mm (%) 74 Poor quality: <8 mm or >13 mm (%) 24
Presence of sCD146 in Embryo Supernatants from Day 2

(36) To confirm the presence of sCD146 in embryos supernatants, inventors performed a western blot analysis on 4 samples with various sCD146 concentration: the first one was evaluated, by ELISA, at 220 pg/ml (transferred at day 2), the second at 1 178 pg/ml (transferred at day 2), the third at 3 850 pg/ml (transferred at day 3) and the last one was the negative control and corresponded to the diluent used for the assay (embryo culture medium, conserved in the same conditions as embryo supernatants but without embryo) (FIG. 2).

(37) Since embryos were transferred at day 2 (D2) or 3 (D3), inventors compared sCD146 concentrations in embryo supernatants at D2 and D3. No significant difference in sCD146 concentrations between D2 or D3 transfers was shown (p=0.36) (FIG. 3).

(38) sCD146 Concentrations and Embryo Quality In practice, embryo selection is based on its morphology. This is the only criterion assessed before transfer. Thereby, inventors studied relationship between concentrations of sCD146 and embryo quality as defined by Istanbul classification. They found that the concentrations of sCD146 did not correlate with the type of embryo (FIG. 4).
sCD146 Concentrations and IVF Outcome

(39) Inventors found a significant difference in sCD146 concentrations between embryos with and without implantation (FIG. 5) with a low concentration associated with high implantation potential. After stepwise multivariable analysis with adjustment to co-variables Istanbul classification and FSH rate, they confirmed the significant association between a low sCD146 concentration and implantation with a Wald chi-square at 5.39 (p=0.02).

(40) Efficacy of sCD146 as a Biomarker of Implantation

(41) The ROC curve computed showed that the optimal sensitivity (71%) and specificity (60%) for implantation was found at 1164 pg/mL sCD146. At this threshold, the percentage of pregnancy was increased by about 40%. This augmentation is maintained into the three Istanbul groups with an augmentation of 20, 30 and 77% in top, fair and poor embryo quality, respectively (FIG. 6).

(42) Conclusions

(43) To avoid the risk of multiple pregnancies and the related complications, it is necessary to select only one embryo to transfer. The prediction of the implantation potential of embryos thus constitutes an imperative in IVF. In this experiment, inventors showed that sCD146 represents an early, noninvasive and innovative biomarker to select the embryo with the highest implantation potential. Interestingly this biomarker is independent of the morphology criteria as defined by Istanbul classification which were up to now the only criteria of selection. Thus the embryo selection with sCD146 can be useful whatever the group of embryos. Therefore sCD146 represents the first biomarker of the embryo selection that improves the accuracy of embryo selection and the effectiveness of IVF.

(44) An early, precise and accurate choice of the embryo with the best potential for implantation indeed constitutes the best strategy to enhance the chances of pregnancy in IVF.

(45) Inventor evidenced in their experiments the presence of sCD146 in embryos supernatants both by ELISA and western blot. This detection could be achieved as soon as day 2 in embryo supernatant. These results bring out sCD146 as an advantageous biomarker in IVF since until now no biomarker could be detected in the blastocyste early stage. Moreover sCD146 use displays another advantage since majority of centers transferred embryos on day 2 or 3, a period which is compatible with sCD146 detection. Finally early detection of sCD146 is also associated with cost reduction.

(46) sCD146 concentration in embryo supernatants represents an early, noninvasive and innovative biomarker to select the embryo with the highest implantation potential. This biomarker advantageously improves the effectiveness of IVF by reducing the time and cost to obtain a pregnancy.

REFERENCES

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