- Email: [email protected]
Human chorionic gonadotropin from day 2 spent embryo culture media and its relationship to embryo development
Human chorionic gonadotropin from day 2 spent embryo culture media and its relationship to embryo development Sivakumar Ramu, Ph.D.,a Brian Acacio, M.D.,b Mark Adamowicz, B.S., T.S. (ABB),b Sylvia Parrett, R.N., B.S.N., M.S.W.,b and Rajasingam S. Jeyendran, D.V.M., Ph.D.a a
Andrology Laboratory Services Inc., Chicago, Illinois; and b Acacio Fertility Center, Laguna Niguel, California
Objective: To detect hCG in spent embryo culture media at day 2 after intracytoplasmic sperm injection and to assess the relationship of hCG to embryo development. Design: Experimental study. Setting: Fertility center and clinical diagnostic laboratory. Sample(s): A total of 102 spent culture media from day 2 human embryos and corresponding unexposed media for blank control. Intervention(s): The culture media samples were tested for hCG by ELISA. Main Outcome Measure(s): Quantity of hCG produced by embryos and correlation with the embryos’ developmental status. Result(s): hCG was found in 93 of 102 culture media tested by enzyme-linked immunosorbent assay. The correlation analysis revealed that the concentration of hCG was independent of embryo developmental status. Conclusion(s): The ability to detect hCG from day 2 spent culture media may be used as a marker for embryo competence. (Fertil Steril 2011;96:615–7. 2011 by American Society for Reproductive Medicine.) Key Words: hCG, embryo development, ELISA, spent culture media
Human chorionic gonadotropin is a glycoprotein hormone comprising 2 subunits, a and b, joined noncovalently (1). It is the most extensively studied hormone of pregnancy and is primarily produced by the embryo and later by the syncytiotrophoblast. HCG appears in maternal blood approximately 6 to 9 days after conception (2). The hormone has been detected at variable levels in culture media of blastocysts (3–6). Although its expression has been documented beginning 2 days after fertilization (5, 7–9), the presence of the hCG molecule has not been reported to date. The objective of our study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) procedure to detect and quantify hCG from day 2 spent embryo culture media and to determine the association of hCG with the developmental potential of the embryo.
MATERIALS AND METHODS A total of 102 day 2 spent embryo culture media from 11 women undergoing intracytoplasmic sperm injection IVF procedures were analyzed for hCG. Institutional Review Board approval was not required for the present study because it does not directly involve any patients or their surgical waste, and the patients gave consent to analyze their spent embryo culture media. Also, the study data were coded and managed in a way that excluded the identification of patients.
Spent Embryo Culture Media Collection Following intracytoplasmic sperm injection, each oocyte was placed into individual droplets of 50 mL of culture medium and incubated. They were exReceived April 19, 2011; revised June 1, 2011; accepted June 14, 2011; published online July 13, 2011. S.R. has nothing to disclose. B.A. has nothing to disclose. M.A. has nothing to disclose. S.P. has nothing to disclose. R.S.J. has nothing to disclose. Reprint requests: Sivakumar Ramu, Ph.D., Andrology Laboratory Services, Andrology Laboratory Services Inc., 680 N. Lake Shore Drive, Suite # 807, Chicago, Illinois 60611 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.06.035
amined for fertilization 16 hours following intracytoplasmic sperm injection. Those having 2 pronuclei were maintained in the same culture media for another 30 to 31 hours. The embryos were then transferred to a different culture media for further embryo development. The spent embryo culture media, in which the embryos were incubated for approximately 48 hours (i.e., until the embryos were transferred), were kept frozen individually in labeled microcentrifuge vials at 20 C until analysis. A sample of pure culture media incubated under the same conditions but without an embryo was also kept frozen and used as a blank control.
hCG Determination by ELISA A quantitative sandwich ELISA was developed in-house and was performed following the optimization on spent embryo culture media. Goat polyclonal antibody to hCG (Abcam) was diluted in coating buffer (0.1 M bicarbonate buffer [pH 9.2]) (Sigma), and polystyrene microtiter plates (Greiner Bio One) were sensitized overnight at 4 C followed by blocking with Trisbuffered saline blocking buffer (Thermo Fisher Scientific) for 1 hour at room temperature. The plates were washed 3 times with phosphatebuffered saline containing 0.05% Tween 20 (Sigma) for 1 hour at room temperature. Synthetic hCG protein standards (Abcam) prepared in appropriate prediluted culture media to avoid matrix difference and samples diluted in phosphate-buffered saline containing 0.05% Tween 20 were pipetted onto the wells and incubated for 1 hour at room temperature. A pure embryo culture medium blank was included in all assays performed. After any unbound compounds were washed away, a mouse monoclonal antibody to hCG-b (Abcam) was added to the wells and incubated for another 1 hour at room temperature. Following a wash, goat anti-mouse IgG conjugated to horseradish peroxidase (Leinco Technologies Inc.) was added to the wells and incubated for 30 minutes at 37 C. SureBlue reserve TMB microwell substrate solution (KPL, Inc.) was added to the wells after the plate was washed with phosphate-buffered saline containing 0.05% Tween 20 and the reaction stopped with 1 M phosphoric acid stop buffer. The optical density of the color developed was measured using ultra microplate reader EL808 (Molecular Devices, Inc.) at 450 nm. The optical density values from the ELISA reader were directly transferred to Microsoft Excel, and the concentrations of the samples were determined.
Fertility and Sterility Vol. 96, No. 3, September 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
615
TABLE 1 hCG concentration in day 2 spent embryo culture media from embryos that were transferred following ICSI IVF procedure and pregnancy outcome among 11 women studied. hCG produced by embryos that were transferred (pg/50 mL) >50.0 25.1–50.0 10.1–25.0 5.5–10.0
P
CC
2.6–5.4 0–2.5
P
C C
P
P
P
P
P
C CC CC
C CC C
CC CC
CC C
CC C
C
NP
NP
B
B
B
NP
B B
NP B B
B B
Note: P ¼ pregnant (n ¼ 7); NP ¼ nonpregnant (n ¼ 4). The bold line represents the 99th percentile (5.5 pg/50 mL; 110 pg/mL; 1 mIU/mL) of accepted levels of hCG. Ramu. hCG in day 2 spent embryo culture media. Fertil Steril 2011.
Reliability of hCG ELISA Procedure Pure embryo culture media spiked with known concentrations of hCG were used to determine the recovery rate of hCG and the reliability of the ELISA procedure. Recovery of hCG was calculated as the concentration determined divided by the expected concentration and expressed as a percentage. To determine interassay and intra-assay variations, hCG spiked culture media were assayed in triplicate on 3 different plates and repeated on at least 3 different days.
Embryo Development Monitoring The embryo culture media were collected on day 2, and embryo quality was monitored on days 3 and 5 based on cleavage rate and morphological features, as previously described (10–13). One or more blastocyst-based morphological characteristics were transferred from the cohort of embryos to all 11 women.
DISCUSSION
Statistical Analysis Pearson correlation and the significance between hCG concentration and embryo development were calculated. P<.05 was considered statistically significant. To calculate the 99% cut-off value for hCG concentrations from the day 2 spent embryo culture media, the results were log transformed for analysis. HCG results are expressed as means SD (range).
RESULTS The reliability of the in-house ELISA was evaluated following optimization, and the recovery of hCG was found to be more than 90%. The coefficient of variation for the intra-assay and interassay was 5.4% and 6.5%, respectively. Of the 102 spent embryo culture media, 9 had no detectable amounts of hCG. However, 7 of the 9 embryos developed into blastocysts. It therefore appears that the failure to detect hCG may be due to technical error rather than embryo quality because we could not rerun these samples and determine the concentrations or find out the cause because of the low sample volumes available for testing. Further, these samples might have had hCG values near or below the lowest detection limit of the assay. Overall, a possibility of more than 1 factor involved in the failed detection of hCG cannot be ruled out. Incidentally, none of these 9 embryos were transferred and hence were excluded from further analysis. The means SD (range) of hCG results per 50 mL for the remaining 93 spent embryo culture media was 16.3 3.6 pg (1.4–225 pg). 616
Ramu et al.
Twenty-two of the 93 spent embryo culture media contained hCG levels less than 3 SD of the mean (99th percentile of accepted level, 5.5 pg/50 mL; 110 pg/mL; 1 mIU/mL) and were considered poor. Eleven of the 22 (50.0%) poor embryos and 16 of 71 (22.5%) acceptable embryos developed to a blastocyst stage. Seven of the 11 patients conceived following 1 or more ETs. The hCG levels produced by these embryos are detailed in Table 1. The hCG levels were not used as selection criteria for ET and were not available at the time of ET in the current study. However, the retrospective analysis revealed that only a single embryo of the 24 embryos transferred had values below the acceptable range in 7 women who conceived, in contrast to 4 of 9 embryos transferred in women who did not conceive; this indicates that hCG levels might be associated with pregnancy outcome.
hCG in day 2 spent embryo culture media
The grading systems based on stage of embryo development to identify embryos with high implantation potential have led to significant improvements in pregnancy rates but have not reduced the number of embryos transferred to either minimize or eliminate twins or higher-order multiple gestations. This has led many investigators to pursue other techniques to assess embryo competence. It has been reported that embryos that result in pregnancy are different in their metabolomic profile compared with embryos that do not (14, 15). Several metabolic parameters using a number of noninvasive techniques on the developing embryos have been measured (14–16). Similarly, other investigators in the past have attempted to evaluate the unique embryo-secreted molecules, such as soluble human leukocyte antigen G from spent embryo culture media, with limited success (17–21). Although each of these markers has demonstrated some usefulness in selecting embryos for transfer, controversy still exists regarding their efficacy. This is probably because not all embryos fail to implant for the same reason. When a single test gives normal results, this does not mean that other defects are not present that may compromise the quality of the embryo. The embryo is complex and becomes compromised when any one of a number of biochemical or morphological entities are disturbed. Normalcy of a single entity or even several entities does not guarantee that the other entities are normal. Unless a large battery of tests are applied and results of each test are graded for their significance in regard to the quality Vol. 96, No. 3, September 2011
of embryo, it will not be possible to state, with any degree of certainty, the reproductive potential of the embryo. Therefore, multiple markers are necessary to identify the one embryo most probable to implant and give rise to a healthy live birth. Although the hCG molecule was the first marker produced by an embryo identified in the spent embryo culture media (3), no further attempts to establish its use as a potential marker of embryo competence has been reported to our knowledge. Its specific function at this stage of embryonic development is currently not known (3, 22, 23). However, recent studies have suggested that the hCG molecule may be involved in the growth and development of the embryoblast at the early stages and implantation at the later stages of development (23). Therefore, we tested day 2 spent embryo culture media collected from 102 embryos and subjected them to ELISA for the quantification of hCG. Our study attempted to establish another marker to be incorporated in the whole gamete of potential markers to assess embryo competence. A poor correlation between hCG levels and embryo development observed in the present study therefore makes the hCG levels more desirable because it indicated that a different and new entity of the embryo is being measured. A high accepted level of hCG (99th percentile) was selected to ensure that the most competent embryo would be identified. This stringent selection resulted in identifying 22 embryos as poor. Of these 22 embryos, 11 (50.0%) developed to the blastocyst stage. Sixteen of 71 (22.5%) embryos identified
as having an acceptable amount of hCG also developed into blastocysts. This may suggest that the embryo’s morphological development is independent of the physiologic development, confirming previously reported findings (4, 6). Also, lack of significant correlation observed between the level of hCG and embryo development on day 2, 3, or 5 (r2 ¼ .001 each; P>.05) in the present study support the above conclusion. Only 9 (8.8%) of the 102 embryos did not yield any hCG results in their spent culture media. However, 7 of the 9 did develop into blastocysts. This may suggest that the failure of hCG detection was due to a technical error. But the possibility that the embryos either did not produce any hCG or produced hCG below the assay detection level cannot be ruled out because hCG production is independent of the morphological development of the embryo. Because more than 1 embryo from a cohort of embryos from the same women was transferred based on the morphological development of the embryo, one could not associate the concentration of hCG with that of the IVF outcome. However, only 1 embryo from a cohort of embryos transferred that resulted in pregnancy had hCG levels lower than 5.5 pg/50 mL (110 pg/mL; 1 mIU/mL) compared with 44.4 % of the embryos that did not yield pregnancy (Table 1). A more detailed prospective study needs to be designed in which many other molecular markers are identified and analyzed simultaneously in the spent embryo culture media to determine the one embryo most probable to implant and give rise to a healthy live birth.
REFERENCES 1. Cole LA. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod Biol Endocrinol 2009;7:8. 2. Edwards RG. Conception in the human female. London: Academic Press; 1980. 3. Fishel SB, Edwards RG, Evans CJ. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 1984;223:816–8. 4. Woodward BJ, Lenton EA, Turner K. Human chorionic gonadotrophin: embryonic secretion is a timedependent phenomenon. Hum Reprod 1993;8:1463–8. 5. Jurisicova A, Antenos M, Kapasi K, Meriano J, Casper RF. Variability in the expression of trophectodermal markers beta-human chorionic gonadotrophin, human leukocyte antigen-G and pregnancy specific beta-1 glycoprotein by the human blastocyst. Hum Reprod 1999;14:1852–8. 6. Lopata A, Oliva K, Stanton PG, Robertson DM. Analysis of chorionic gonadotrophin secreted by cultured human blastocysts. Mol Hum Reprod 1997;3:517–21. 7. Bonduelle ML, Dodd R, Liebaers I, Van Steirteghem A, Williamson R, Akhurst R. Chorionic gonadotrophin-beta mRNA, a trophoblast marker, is expressed in human 8-cell embryos derived from tripronucleate zygotes. Hum Reprod 1988;3:909–14. 8. Adjaye J, Bolton V, Monk M. Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. Gene 1999;237:373–83. 9. Hansis C, Grifo JA, Tang Y, Krey LC. Assessment of beta-HCG, beta-LH mRNA and ploidy in individual human blastomeres. Reprod Biomed Online 2002;5: 156–61.
Fertility and Sterility
10. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001;76:1175–80. 11. Van Royen E, Mangelschots K, De Neubourg D, Valkenburg M, Van de Meerssche M, Ryckaert G, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999;14:2345–9. 12. Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol 1999;11:307–11. 13. Sjoblom P, Menezes J, Cummins L, Mathiyalagan B, Costello MF. Prediction of embryo developmental potential and pregnancy based on early stage morphological characteristics. Fertil Steril 2006;86: 848–61. 14. Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007;88:1350–7. 15. Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod 2008;14:679–90. 16. Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2008;90:2183–9. 17. Sher G, Keskintepe L, Nouriani M, Roussev R, Batzofin J. Expression of sHLA-G in supernatants
18.
19.
20.
21.
22.
23.
of individually cultured 46-h embryos: a potentially valuable indicator of ‘embryo competency’ and IVF outcome. Reprod Biomed Online 2004;9:74–8. Rebmann V, Switala M, Eue I, Schwahn E, Merzenich M, Grosse-Wilde H. Rapid evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos. Hum Immunol 2007;68:251–8. Noci I, Fuzzi B, Rizzo R, Melchiorri L, Criscuoli L, Dabizzi S, et al. Embryonic soluble HLA-G as a marker of developmental potential in embryos. Hum Reprod 2005;20:138–46. Vercammen MJ, Verloes A, Van de Velde H, Haentjens P. Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment: meta-analysis. Hum Reprod Update 2008;14:209–18. Tabiasco J, Perrier d’Hauterive S, Thonon F, Parinaud J, Leandri R, Foidart JM, et al. Soluble HLA-G in IVF/ICSI embryo culture supernatants does not always predict implantation success: a multicentre study. Reprod Biomed Online 2009;18: 374–81. Edwards RG, Hansis C. Initial differentiation of blastomeres in 4-cell human embryos and its significance for early embryogenesis and implantation. Reprod Biomed Online 2005;11:206–18. Gallego MJ, Porayette P, Kaltcheva MM, Bowen RL, Vadakkadath Meethal S, Atwood CS. The pregnancy hormones human chorionic gonadotropin and progesterone induce human embryonic stem cell proliferation and differentiation into neuroectodermal rosettes. Stem Cell Res Ther 2010;1:28.
617
Andrology Laboratory Services Inc., Chicago, Illinois; and b Acacio Fertility Center, Laguna Niguel, California
Objective: To detect hCG in spent embryo culture media at day 2 after intracytoplasmic sperm injection and to assess the relationship of hCG to embryo development. Design: Experimental study. Setting: Fertility center and clinical diagnostic laboratory. Sample(s): A total of 102 spent culture media from day 2 human embryos and corresponding unexposed media for blank control. Intervention(s): The culture media samples were tested for hCG by ELISA. Main Outcome Measure(s): Quantity of hCG produced by embryos and correlation with the embryos’ developmental status. Result(s): hCG was found in 93 of 102 culture media tested by enzyme-linked immunosorbent assay. The correlation analysis revealed that the concentration of hCG was independent of embryo developmental status. Conclusion(s): The ability to detect hCG from day 2 spent culture media may be used as a marker for embryo competence. (Fertil Steril 2011;96:615–7. 2011 by American Society for Reproductive Medicine.) Key Words: hCG, embryo development, ELISA, spent culture media
Human chorionic gonadotropin is a glycoprotein hormone comprising 2 subunits, a and b, joined noncovalently (1). It is the most extensively studied hormone of pregnancy and is primarily produced by the embryo and later by the syncytiotrophoblast. HCG appears in maternal blood approximately 6 to 9 days after conception (2). The hormone has been detected at variable levels in culture media of blastocysts (3–6). Although its expression has been documented beginning 2 days after fertilization (5, 7–9), the presence of the hCG molecule has not been reported to date. The objective of our study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) procedure to detect and quantify hCG from day 2 spent embryo culture media and to determine the association of hCG with the developmental potential of the embryo.
MATERIALS AND METHODS A total of 102 day 2 spent embryo culture media from 11 women undergoing intracytoplasmic sperm injection IVF procedures were analyzed for hCG. Institutional Review Board approval was not required for the present study because it does not directly involve any patients or their surgical waste, and the patients gave consent to analyze their spent embryo culture media. Also, the study data were coded and managed in a way that excluded the identification of patients.
Spent Embryo Culture Media Collection Following intracytoplasmic sperm injection, each oocyte was placed into individual droplets of 50 mL of culture medium and incubated. They were exReceived April 19, 2011; revised June 1, 2011; accepted June 14, 2011; published online July 13, 2011. S.R. has nothing to disclose. B.A. has nothing to disclose. M.A. has nothing to disclose. S.P. has nothing to disclose. R.S.J. has nothing to disclose. Reprint requests: Sivakumar Ramu, Ph.D., Andrology Laboratory Services, Andrology Laboratory Services Inc., 680 N. Lake Shore Drive, Suite # 807, Chicago, Illinois 60611 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.06.035
amined for fertilization 16 hours following intracytoplasmic sperm injection. Those having 2 pronuclei were maintained in the same culture media for another 30 to 31 hours. The embryos were then transferred to a different culture media for further embryo development. The spent embryo culture media, in which the embryos were incubated for approximately 48 hours (i.e., until the embryos were transferred), were kept frozen individually in labeled microcentrifuge vials at 20 C until analysis. A sample of pure culture media incubated under the same conditions but without an embryo was also kept frozen and used as a blank control.
hCG Determination by ELISA A quantitative sandwich ELISA was developed in-house and was performed following the optimization on spent embryo culture media. Goat polyclonal antibody to hCG (Abcam) was diluted in coating buffer (0.1 M bicarbonate buffer [pH 9.2]) (Sigma), and polystyrene microtiter plates (Greiner Bio One) were sensitized overnight at 4 C followed by blocking with Trisbuffered saline blocking buffer (Thermo Fisher Scientific) for 1 hour at room temperature. The plates were washed 3 times with phosphatebuffered saline containing 0.05% Tween 20 (Sigma) for 1 hour at room temperature. Synthetic hCG protein standards (Abcam) prepared in appropriate prediluted culture media to avoid matrix difference and samples diluted in phosphate-buffered saline containing 0.05% Tween 20 were pipetted onto the wells and incubated for 1 hour at room temperature. A pure embryo culture medium blank was included in all assays performed. After any unbound compounds were washed away, a mouse monoclonal antibody to hCG-b (Abcam) was added to the wells and incubated for another 1 hour at room temperature. Following a wash, goat anti-mouse IgG conjugated to horseradish peroxidase (Leinco Technologies Inc.) was added to the wells and incubated for 30 minutes at 37 C. SureBlue reserve TMB microwell substrate solution (KPL, Inc.) was added to the wells after the plate was washed with phosphate-buffered saline containing 0.05% Tween 20 and the reaction stopped with 1 M phosphoric acid stop buffer. The optical density of the color developed was measured using ultra microplate reader EL808 (Molecular Devices, Inc.) at 450 nm. The optical density values from the ELISA reader were directly transferred to Microsoft Excel, and the concentrations of the samples were determined.
Fertility and Sterility Vol. 96, No. 3, September 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
615
TABLE 1 hCG concentration in day 2 spent embryo culture media from embryos that were transferred following ICSI IVF procedure and pregnancy outcome among 11 women studied. hCG produced by embryos that were transferred (pg/50 mL) >50.0 25.1–50.0 10.1–25.0 5.5–10.0
P
CC
2.6–5.4 0–2.5
P
C C
P
P
P
P
P
C CC CC
C CC C
CC CC
CC C
CC C
C
NP
NP
B
B
B
NP
B B
NP B B
B B
Note: P ¼ pregnant (n ¼ 7); NP ¼ nonpregnant (n ¼ 4). The bold line represents the 99th percentile (5.5 pg/50 mL; 110 pg/mL; 1 mIU/mL) of accepted levels of hCG. Ramu. hCG in day 2 spent embryo culture media. Fertil Steril 2011.
Reliability of hCG ELISA Procedure Pure embryo culture media spiked with known concentrations of hCG were used to determine the recovery rate of hCG and the reliability of the ELISA procedure. Recovery of hCG was calculated as the concentration determined divided by the expected concentration and expressed as a percentage. To determine interassay and intra-assay variations, hCG spiked culture media were assayed in triplicate on 3 different plates and repeated on at least 3 different days.
Embryo Development Monitoring The embryo culture media were collected on day 2, and embryo quality was monitored on days 3 and 5 based on cleavage rate and morphological features, as previously described (10–13). One or more blastocyst-based morphological characteristics were transferred from the cohort of embryos to all 11 women.
DISCUSSION
Statistical Analysis Pearson correlation and the significance between hCG concentration and embryo development were calculated. P<.05 was considered statistically significant. To calculate the 99% cut-off value for hCG concentrations from the day 2 spent embryo culture media, the results were log transformed for analysis. HCG results are expressed as means SD (range).
RESULTS The reliability of the in-house ELISA was evaluated following optimization, and the recovery of hCG was found to be more than 90%. The coefficient of variation for the intra-assay and interassay was 5.4% and 6.5%, respectively. Of the 102 spent embryo culture media, 9 had no detectable amounts of hCG. However, 7 of the 9 embryos developed into blastocysts. It therefore appears that the failure to detect hCG may be due to technical error rather than embryo quality because we could not rerun these samples and determine the concentrations or find out the cause because of the low sample volumes available for testing. Further, these samples might have had hCG values near or below the lowest detection limit of the assay. Overall, a possibility of more than 1 factor involved in the failed detection of hCG cannot be ruled out. Incidentally, none of these 9 embryos were transferred and hence were excluded from further analysis. The means SD (range) of hCG results per 50 mL for the remaining 93 spent embryo culture media was 16.3 3.6 pg (1.4–225 pg). 616
Ramu et al.
Twenty-two of the 93 spent embryo culture media contained hCG levels less than 3 SD of the mean (99th percentile of accepted level, 5.5 pg/50 mL; 110 pg/mL; 1 mIU/mL) and were considered poor. Eleven of the 22 (50.0%) poor embryos and 16 of 71 (22.5%) acceptable embryos developed to a blastocyst stage. Seven of the 11 patients conceived following 1 or more ETs. The hCG levels produced by these embryos are detailed in Table 1. The hCG levels were not used as selection criteria for ET and were not available at the time of ET in the current study. However, the retrospective analysis revealed that only a single embryo of the 24 embryos transferred had values below the acceptable range in 7 women who conceived, in contrast to 4 of 9 embryos transferred in women who did not conceive; this indicates that hCG levels might be associated with pregnancy outcome.
hCG in day 2 spent embryo culture media
The grading systems based on stage of embryo development to identify embryos with high implantation potential have led to significant improvements in pregnancy rates but have not reduced the number of embryos transferred to either minimize or eliminate twins or higher-order multiple gestations. This has led many investigators to pursue other techniques to assess embryo competence. It has been reported that embryos that result in pregnancy are different in their metabolomic profile compared with embryos that do not (14, 15). Several metabolic parameters using a number of noninvasive techniques on the developing embryos have been measured (14–16). Similarly, other investigators in the past have attempted to evaluate the unique embryo-secreted molecules, such as soluble human leukocyte antigen G from spent embryo culture media, with limited success (17–21). Although each of these markers has demonstrated some usefulness in selecting embryos for transfer, controversy still exists regarding their efficacy. This is probably because not all embryos fail to implant for the same reason. When a single test gives normal results, this does not mean that other defects are not present that may compromise the quality of the embryo. The embryo is complex and becomes compromised when any one of a number of biochemical or morphological entities are disturbed. Normalcy of a single entity or even several entities does not guarantee that the other entities are normal. Unless a large battery of tests are applied and results of each test are graded for their significance in regard to the quality Vol. 96, No. 3, September 2011
of embryo, it will not be possible to state, with any degree of certainty, the reproductive potential of the embryo. Therefore, multiple markers are necessary to identify the one embryo most probable to implant and give rise to a healthy live birth. Although the hCG molecule was the first marker produced by an embryo identified in the spent embryo culture media (3), no further attempts to establish its use as a potential marker of embryo competence has been reported to our knowledge. Its specific function at this stage of embryonic development is currently not known (3, 22, 23). However, recent studies have suggested that the hCG molecule may be involved in the growth and development of the embryoblast at the early stages and implantation at the later stages of development (23). Therefore, we tested day 2 spent embryo culture media collected from 102 embryos and subjected them to ELISA for the quantification of hCG. Our study attempted to establish another marker to be incorporated in the whole gamete of potential markers to assess embryo competence. A poor correlation between hCG levels and embryo development observed in the present study therefore makes the hCG levels more desirable because it indicated that a different and new entity of the embryo is being measured. A high accepted level of hCG (99th percentile) was selected to ensure that the most competent embryo would be identified. This stringent selection resulted in identifying 22 embryos as poor. Of these 22 embryos, 11 (50.0%) developed to the blastocyst stage. Sixteen of 71 (22.5%) embryos identified
as having an acceptable amount of hCG also developed into blastocysts. This may suggest that the embryo’s morphological development is independent of the physiologic development, confirming previously reported findings (4, 6). Also, lack of significant correlation observed between the level of hCG and embryo development on day 2, 3, or 5 (r2 ¼ .001 each; P>.05) in the present study support the above conclusion. Only 9 (8.8%) of the 102 embryos did not yield any hCG results in their spent culture media. However, 7 of the 9 did develop into blastocysts. This may suggest that the failure of hCG detection was due to a technical error. But the possibility that the embryos either did not produce any hCG or produced hCG below the assay detection level cannot be ruled out because hCG production is independent of the morphological development of the embryo. Because more than 1 embryo from a cohort of embryos from the same women was transferred based on the morphological development of the embryo, one could not associate the concentration of hCG with that of the IVF outcome. However, only 1 embryo from a cohort of embryos transferred that resulted in pregnancy had hCG levels lower than 5.5 pg/50 mL (110 pg/mL; 1 mIU/mL) compared with 44.4 % of the embryos that did not yield pregnancy (Table 1). A more detailed prospective study needs to be designed in which many other molecular markers are identified and analyzed simultaneously in the spent embryo culture media to determine the one embryo most probable to implant and give rise to a healthy live birth.
REFERENCES 1. Cole LA. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod Biol Endocrinol 2009;7:8. 2. Edwards RG. Conception in the human female. London: Academic Press; 1980. 3. Fishel SB, Edwards RG, Evans CJ. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 1984;223:816–8. 4. Woodward BJ, Lenton EA, Turner K. Human chorionic gonadotrophin: embryonic secretion is a timedependent phenomenon. Hum Reprod 1993;8:1463–8. 5. Jurisicova A, Antenos M, Kapasi K, Meriano J, Casper RF. Variability in the expression of trophectodermal markers beta-human chorionic gonadotrophin, human leukocyte antigen-G and pregnancy specific beta-1 glycoprotein by the human blastocyst. Hum Reprod 1999;14:1852–8. 6. Lopata A, Oliva K, Stanton PG, Robertson DM. Analysis of chorionic gonadotrophin secreted by cultured human blastocysts. Mol Hum Reprod 1997;3:517–21. 7. Bonduelle ML, Dodd R, Liebaers I, Van Steirteghem A, Williamson R, Akhurst R. Chorionic gonadotrophin-beta mRNA, a trophoblast marker, is expressed in human 8-cell embryos derived from tripronucleate zygotes. Hum Reprod 1988;3:909–14. 8. Adjaye J, Bolton V, Monk M. Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. Gene 1999;237:373–83. 9. Hansis C, Grifo JA, Tang Y, Krey LC. Assessment of beta-HCG, beta-LH mRNA and ploidy in individual human blastomeres. Reprod Biomed Online 2002;5: 156–61.
Fertility and Sterility
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