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Purification of Human Chorionic Gonadotropin (hCG) for Intrauterine Injection
a
University of California, Los Angeles, 757 Westwood Plaza, Los Angeles, CA 90095, USA; bSouthern California Reproductive Center, 450 Roxbury Drive, Suite 500, Beverly Hills, CA 91210, USA.
POSTER PRESENTATIONS
P-1 Purification of Human Chorionic Gonadotropin (hCG) for Intrauterine Injection. A. M. Rosen,a M. C. Hermoso,b H. Cook-Andersen,c D. de Ziegler,d D. R. Meldrum.b aAlbany Medical College, Albany, NY, USA; bReproductive Partners Medical Group, Redondo Beach, CA, USA; cDepartment of Reproductive Medicine, University of California San Diego, San Diego, CA, USA; dDepartment of Reproductive Endocrinology and Infertility, Universite Paris Descartes, Paris, France. BACKGROUND: Recent research showed that intrauterine injection of hCG before embryo transfer resulted in significantly increased embryo implantation. (1) Purification of hCG may allow for maximal benefit. OBJECTIVE(S): The purpose of this study was to evaluate a filtration protocol that would retain hCG concentration and bioactivity while allowing for improved mouse embryo development to the blastocyst stage when compared with unfiltered preparations. MATERIALS AND METHOD(S): A simple and inexpensive size-exclusion membrane was used to purify hCG and the product was evaluated using in vitro and in vivo assays. HCG was dissolved at 500 IU per 40 uL in Vitrolife G2 Plus, placed in an Amicon Ultra-15 (3K) filter, diluted 20-fold with G-Rinse, centrifuged for 1 hour at 4,000 RPM, and reconstituted in G2 Plus. Bioactivity was assessed by injecting 10 IU of hCG into female mice to induce ovulation in vivo. Two-cell mouse embryos were cultured in media with 500 IU per 40 uL of filtered or unfiltered hCG to test viability of the embryos. Osmolarity, pH, and hCG concentration of the preparations were also measured. RESULT(S): Before purification, hCG arrested growth of mouse embryos at the cleavage stage. Purification of hCG resulted in marked improvements in blastocyst development (from 0% to >90%). HCG retained bioactivity after purification as assessed by stimulation of ovulation in mice. After purification, improvements in osmolarity (from 431 to 293 milliosmoles/L) and pH (from 6.7 to 7.4) were observed. Over 92% of the hCG was recovered after purification (range 85-109%). Testing of vial rinse showed high embryo viability. TABLE. (mean of similar values using 10 vials from three manufacturers; 1. Unfiltered 2. Filtered)
1 2
Osmolarity (mOsm/kg)
hCG conc (mIU/ml)
431 293
103,797 95,629 (92% recovery)
Toxicity Assay: cleaved mouse Bioactivity Assay: number of fertilized embryos reaching blastocyst (%) oocytes/mouse 15.4 16.2
0/60 ¼ 0% 84/90 ¼ 93%
The pH before and after processing was estimated at 6.7 and 7.4 respectively. After removing the reconstituted hCG from the vial, a further rinse of the vial with media failed to detect any embryo toxicity using one-cell mouse embryos (% blast¼ 100). CONCLUSION(S): While hCG reconstituted in media was highly toxic to mouse embryos, purified hCG allowed a high rate of blastocyst development. Embryo toxicity was due to additional solutes in the hCG powder and possibly to contaminants introduced during processing, but not to toxicity of the vial or the rubber stopper. This simple and inexpensive technique to purify hCG should further improve the already demonstrated enhancement of embryo implantation with intrauterine injection. SUPPORT: None. Reference 1. Mansour R, Tawab N, Kamal O, El-Faissal Y, Serour A, Aboulghar M, Serour G. Intrauterine injection of human chorionic gonadotropin before embryo transfer significantly improves the implantation and pregnancy rates in in vitro fertilization/intracytoplasmic sperm injection: a prospective randomized study. Fertil Steril 2011;96:1370–4.
P-2 The Relationship between Gender and Aneuploidy Rates in Patients Undergoing In Vitro Fertilization (IVF) for Sex Selection. Alin L. Akopians, M.D., Ph.D.,a Gayane Ambartsumyan, M.D., Ph.D.,a Mark Surrey, M.D.,b Christine Briton-Jones, Ph.D.,b David Hill, Ph.D.b
S8
PCRS Abstracts
BACKGROUND: Recent literature suggests possible gender specific differences in aneuploidy in patients undergoing preimplantation genetic screening (PGS). No data exists on gender specific aneuploidy in patients undergoing PGS for sex selection. OBJECTIVE(S): To assess the relationship between embryo gender and aneuploidy rates in patients undergoing IVF for sex selection. In addition, to evaluate gender specific differences in blastulation rates (BR) and aneuploid blastocyst rates (ABR) in sex-selected groups. DESIGN: Historical cohort study. MATERIALS AND METHOD(S): A total of 846 embryos from 97 IVF cycles undergoing sex selection were studied. Patients undergoing PGS with both microarray-based comparative genomic hybridization (aCGH) and fluorescent in-situ hybridization (FISH) were included. There were 467 (55%) embryos in female sex-selected group and 379 (45%) in male sex-selected group. Embryo biopsies were performed on day 3. Aneuploidy rates (AR), blastulation rates (BR), and aneuploid blastocyst rates (ABR) were calculated for each gender. AR was the ratio of abnormal embryos per total embryos biopsied for each gender. BR was the ratio of total blastocysts per day 3 embryos. ABR was the ratio of abnormal blastocysts per total blastocysts for each gender. The logit of the rates were then compared between genders for each sex-selected group using paired t-test. RESULT(S): For female sex-selected group, there was no difference in overall male and female embryos (0.48 v 0.52), yet more male embryos were noted in male sex selection group (0.55 v 0.45, p¼0.04). For patients undergoing female sex selection, there was no statistically significant difference in female or male aneuploidy, blastulation, and aneuploid blastocyst rates (Table 1). Similar results were noted in the male sex-selected group (Table 1). Interestingly, both sex-selected groups demonstrated a 10% increase in aneuploid blastocyst rates in the corresponding gender being selected; however the difference was not statistically significant. CONCLUSION(S): Our study suggests that the rates of whole chromosomal abnormalities are not different between genders in patients undergoing IVF for sex selection. One still cannot rule out, however, the contribution of other genetic abnormalities not detected by our PGS technology. Larger populations are needed to further evaluate the significance of the gender-specific 10% increase in aneuploid blastocysts seen in our study. SUPPORT: None.
TABLE 1. Summary of gender differences in both male and female sex selection groups
Female Sex selection
Male embryo
Female embryo
p value
Embryo sex 225 (225/467¼0.48) 242 (242/467¼0.52) 0.43 (number) Aneuploidy 0.42 0.49 0.45 rate (AR) Blastulation Rate 0.26 0.23 0.44 % abnormal blast 0.36 0.46 0.32 Male Sex Selection
Male embryo
Female embryo
Embryo sex 209 (209/379¼0.55) 170 (170/379¼0.45) Abnormal rate 0.52 0.55 Blastulation rate 0.32 0.25 % abn blast 0.49 0.4
P value 0.04 0.7 0.22 0.41
P-3 Egg Donor Informed Consent Tool (EDICT): Oocyte Donors’ Understanding as Assessed by a Novel Valid Informed Consent Tool. A. Skillern, M. Cedars, L. Pasch, K. Forsberg, H. Huddleston. Dept. of OB/GYN and Reproductive Sciences, University of California, San Francisco, CA, USA. BACKGROUND: The concept of informed consent has been described in the bioethics literature as a process comprised of three elements: disclosure, capacity, and voluntariness (1). ‘‘Disclosure" is defined as relevant
Vol. 99, No. 3, Supplement, March 1, 2013
University of California, Los Angeles, 757 Westwood Plaza, Los Angeles, CA 90095, USA; bSouthern California Reproductive Center, 450 Roxbury Drive, Suite 500, Beverly Hills, CA 91210, USA.
POSTER PRESENTATIONS
P-1 Purification of Human Chorionic Gonadotropin (hCG) for Intrauterine Injection. A. M. Rosen,a M. C. Hermoso,b H. Cook-Andersen,c D. de Ziegler,d D. R. Meldrum.b aAlbany Medical College, Albany, NY, USA; bReproductive Partners Medical Group, Redondo Beach, CA, USA; cDepartment of Reproductive Medicine, University of California San Diego, San Diego, CA, USA; dDepartment of Reproductive Endocrinology and Infertility, Universite Paris Descartes, Paris, France. BACKGROUND: Recent research showed that intrauterine injection of hCG before embryo transfer resulted in significantly increased embryo implantation. (1) Purification of hCG may allow for maximal benefit. OBJECTIVE(S): The purpose of this study was to evaluate a filtration protocol that would retain hCG concentration and bioactivity while allowing for improved mouse embryo development to the blastocyst stage when compared with unfiltered preparations. MATERIALS AND METHOD(S): A simple and inexpensive size-exclusion membrane was used to purify hCG and the product was evaluated using in vitro and in vivo assays. HCG was dissolved at 500 IU per 40 uL in Vitrolife G2 Plus, placed in an Amicon Ultra-15 (3K) filter, diluted 20-fold with G-Rinse, centrifuged for 1 hour at 4,000 RPM, and reconstituted in G2 Plus. Bioactivity was assessed by injecting 10 IU of hCG into female mice to induce ovulation in vivo. Two-cell mouse embryos were cultured in media with 500 IU per 40 uL of filtered or unfiltered hCG to test viability of the embryos. Osmolarity, pH, and hCG concentration of the preparations were also measured. RESULT(S): Before purification, hCG arrested growth of mouse embryos at the cleavage stage. Purification of hCG resulted in marked improvements in blastocyst development (from 0% to >90%). HCG retained bioactivity after purification as assessed by stimulation of ovulation in mice. After purification, improvements in osmolarity (from 431 to 293 milliosmoles/L) and pH (from 6.7 to 7.4) were observed. Over 92% of the hCG was recovered after purification (range 85-109%). Testing of vial rinse showed high embryo viability. TABLE. (mean of similar values using 10 vials from three manufacturers; 1. Unfiltered 2. Filtered)
1 2
Osmolarity (mOsm/kg)
hCG conc (mIU/ml)
431 293
103,797 95,629 (92% recovery)
Toxicity Assay: cleaved mouse Bioactivity Assay: number of fertilized embryos reaching blastocyst (%) oocytes/mouse 15.4 16.2
0/60 ¼ 0% 84/90 ¼ 93%
The pH before and after processing was estimated at 6.7 and 7.4 respectively. After removing the reconstituted hCG from the vial, a further rinse of the vial with media failed to detect any embryo toxicity using one-cell mouse embryos (% blast¼ 100). CONCLUSION(S): While hCG reconstituted in media was highly toxic to mouse embryos, purified hCG allowed a high rate of blastocyst development. Embryo toxicity was due to additional solutes in the hCG powder and possibly to contaminants introduced during processing, but not to toxicity of the vial or the rubber stopper. This simple and inexpensive technique to purify hCG should further improve the already demonstrated enhancement of embryo implantation with intrauterine injection. SUPPORT: None. Reference 1. Mansour R, Tawab N, Kamal O, El-Faissal Y, Serour A, Aboulghar M, Serour G. Intrauterine injection of human chorionic gonadotropin before embryo transfer significantly improves the implantation and pregnancy rates in in vitro fertilization/intracytoplasmic sperm injection: a prospective randomized study. Fertil Steril 2011;96:1370–4.
P-2 The Relationship between Gender and Aneuploidy Rates in Patients Undergoing In Vitro Fertilization (IVF) for Sex Selection. Alin L. Akopians, M.D., Ph.D.,a Gayane Ambartsumyan, M.D., Ph.D.,a Mark Surrey, M.D.,b Christine Briton-Jones, Ph.D.,b David Hill, Ph.D.b
S8
PCRS Abstracts
BACKGROUND: Recent literature suggests possible gender specific differences in aneuploidy in patients undergoing preimplantation genetic screening (PGS). No data exists on gender specific aneuploidy in patients undergoing PGS for sex selection. OBJECTIVE(S): To assess the relationship between embryo gender and aneuploidy rates in patients undergoing IVF for sex selection. In addition, to evaluate gender specific differences in blastulation rates (BR) and aneuploid blastocyst rates (ABR) in sex-selected groups. DESIGN: Historical cohort study. MATERIALS AND METHOD(S): A total of 846 embryos from 97 IVF cycles undergoing sex selection were studied. Patients undergoing PGS with both microarray-based comparative genomic hybridization (aCGH) and fluorescent in-situ hybridization (FISH) were included. There were 467 (55%) embryos in female sex-selected group and 379 (45%) in male sex-selected group. Embryo biopsies were performed on day 3. Aneuploidy rates (AR), blastulation rates (BR), and aneuploid blastocyst rates (ABR) were calculated for each gender. AR was the ratio of abnormal embryos per total embryos biopsied for each gender. BR was the ratio of total blastocysts per day 3 embryos. ABR was the ratio of abnormal blastocysts per total blastocysts for each gender. The logit of the rates were then compared between genders for each sex-selected group using paired t-test. RESULT(S): For female sex-selected group, there was no difference in overall male and female embryos (0.48 v 0.52), yet more male embryos were noted in male sex selection group (0.55 v 0.45, p¼0.04). For patients undergoing female sex selection, there was no statistically significant difference in female or male aneuploidy, blastulation, and aneuploid blastocyst rates (Table 1). Similar results were noted in the male sex-selected group (Table 1). Interestingly, both sex-selected groups demonstrated a 10% increase in aneuploid blastocyst rates in the corresponding gender being selected; however the difference was not statistically significant. CONCLUSION(S): Our study suggests that the rates of whole chromosomal abnormalities are not different between genders in patients undergoing IVF for sex selection. One still cannot rule out, however, the contribution of other genetic abnormalities not detected by our PGS technology. Larger populations are needed to further evaluate the significance of the gender-specific 10% increase in aneuploid blastocysts seen in our study. SUPPORT: None.
TABLE 1. Summary of gender differences in both male and female sex selection groups
Female Sex selection
Male embryo
Female embryo
p value
Embryo sex 225 (225/467¼0.48) 242 (242/467¼0.52) 0.43 (number) Aneuploidy 0.42 0.49 0.45 rate (AR) Blastulation Rate 0.26 0.23 0.44 % abnormal blast 0.36 0.46 0.32 Male Sex Selection
Male embryo
Female embryo
Embryo sex 209 (209/379¼0.55) 170 (170/379¼0.45) Abnormal rate 0.52 0.55 Blastulation rate 0.32 0.25 % abn blast 0.49 0.4
P value 0.04 0.7 0.22 0.41
P-3 Egg Donor Informed Consent Tool (EDICT): Oocyte Donors’ Understanding as Assessed by a Novel Valid Informed Consent Tool. A. Skillern, M. Cedars, L. Pasch, K. Forsberg, H. Huddleston. Dept. of OB/GYN and Reproductive Sciences, University of California, San Francisco, CA, USA. BACKGROUND: The concept of informed consent has been described in the bioethics literature as a process comprised of three elements: disclosure, capacity, and voluntariness (1). ‘‘Disclosure" is defined as relevant
Vol. 99, No. 3, Supplement, March 1, 2013