Laser-assisted hatching (LAH) of cryopreserved embryos – the significance of hole size

OBJECTIVE: Criteria for poor responders include advanced age, previous cycle cancellation, or an abnormal ovarian reserve. The strategy proposed to improve the outcome of poor responders was growth hormone(GH). In order to verify the advantage of GH administration, we conducted a study to investigate the efficacy of co-treatment with GH in control ovarian stimulation(COS) of poor responders undergoing IVF/ET. DESIGN: Retrospective case control study in an university hospital. MATERIALS AND METHODS: 184 patients who matched the criteria for poor responders were included. The criteria was either advanced age (>40y/o), previous oocyte retrieval <3, or low AMH level(<1 ng/ml).94 couples were allocated to GH co-treatment group(group A), the other 90 served as the control(group B). Group A received a GnRHa ultra-long protocol with Leuprolide Acetate 1.88mg started in day3 of preceding cycle.Ovulation induction with gonadotropin since day 35-40. GH co-treatment was administrated daily with 4IU for three days and adjusted according by until hCG day. The patients in group B received the same treatment without GH. RESULTS: The co-treatment with GH displayed a favorable COS and pregnancy rate. The E2 level (mean
SE)on hCG day in Group A was significantly higher than Group B (679
47 vs 457
38 pg/ml; P<0.001). The oocyte retrieved in Group A and Group B was 5.5
0.3 and 2.1
0.1.(P<0.001) Number of embryo transfer was also higher in Group A (2.6
0.1vs1.7
0.1; P<0.001). Pregnancy rate in Group A was statistically higher than groupB (31.9%vs16.7%; P<0.05). However, there was no difference in abortion rate between these two groups. CONCLUSION: Growth hormone is reported to modulate the action of FSH on granulosa cells by up-regulating the local synthesis of IGF-1,and it may also involve the direct action on follicular maturation. From our study, co-treatment with GH in GnRHa ultralong protocol seems to achieve a satisfactory E2 levels, oocyte numbers, embryo numbers as well as pregnancy outcome, especially in poor responders of IVF patients.

PROCEDURES AND TECHNIQUES-LABORATORY: ART O-265 Wednesday, October 24, 2012 11:15 AM DENSITY GRADIENT CENTRIFUGATION PRIOR TO CRYOPRESERVATION AND ANTIOXYDANT SUPPLEMENTATION BY HYPOTAURINE IMPROVE POST-THAW SPERM QUALITY OF INFERTILE MEN WITH OLIGOASTHENOTERATOZOOSPER MIA. F. Brugnon,a,b L. Ouchchane,c,d,e C. Artonne,b H. Pons-Rejraji,a,b L. Janny.a,b aBiologie de la Reproduction, AMP, CECOS, CHU Clermont Ferrand, CHU Estaing, Clermont Ferrand, France; bG
en
etique Reproduction D
eveloppement,GReD, Universit
e d’Auvergne, INSERM U1103, CNRS UMR 6393, Facult
e de M
edecine, Clermont Ferrand, France; cP^ole Sant
e Publique, M
edecine L
egale, Qualit
e, Vigilances, Unit
e de Biostatistique, Informatique M
edicale et Technologies de Communication, CHU Clermont Ferrand, Clermont Ferrand, France; dLaboratoire de Biostatistique, Informatique M
edicale et Technologies de Communication, Universit
e d’Auvergne, Clermont Ferrand, France; eISIT, UMR6284, Universit
e d’Auvergne, Clermont Ferrand, France. OBJECTIVE: To analyze the effects of the sperm selection before or after freezing by density gradient centrifugation and antioxidant supplementation by hypotaurine in sperm samples of patients with oligoasthenoteratozoospermia. DESIGN: Retrospective study. MATERIALS AND METHODS: Four procedures were compared in oligoasthenoteratozoospermia sperm samples (n¼16 for each procedure). A: Sperm freezing before selection without hypotaurine supplementation; B: Sperm freezing before selection with hypotaurine supplementation; C: Sperm selection before freezing without hypotaurine supplementation; D: Sperm selection before freezing with hypotaurine supplementation. The quality of recovered spermatozoa was assessed measuring markers of apoptosis (mitochondrial membrane potential (DJm), DNA fragmentation (TUNEL), externalisation of plasma membrane phosphatidylserine (PS)). RESULTS: The percentage of DJ high spermatozoa was higher (P¼0.001) when the selection was performed before freezing compared with the procedures whose selection was performed after sperm freezing with (D vs. B: 58.09
14.02% vs. 46.69
21.92%) or without (C vs. A: 57.00
20.70% vs. 35.36
19.97 %) hypotaurine supplementation. The percentage of TUNEL+ spermatozoa was significantly lower (P¼0.001) when the selection was performed before freezing compared with the procedures whose selection was performed after sperm freezing with (D vs. B: 38.59
25.37% vs. 55.68
20.37%) or without hypotaurine supplementation (C vs. A: 37.21
23.72% vs. 70.99
20.40%). The percentage of live spermatozoa with PS externalization was lower (P¼0.003) when the selection was per-

FERTILITY & STERILITYÒ

formed before freezing with hypotaurine supplementation (D: 1.85
1.22%) compared with the same sequence without hypotaurine supplementation (C: 3.36
2.09%). CONCLUSION: Density gradient prior to cryopreservation and hypotaurine supplementation is the best sperm procedure providing the lower expression of apoptosis markers in spermatozoa of infertile men with oligoasthenoteratozoospermia. Supported by: Hospital grant for clinical research. O-266 Wednesday, October 24, 2012 11:30 AM LIVE-CELL INTRA-OOCYTE LIPID ANALYSIS AND QUANTIFICATION WITH HYPERSPECTRAL IMAGING BY MULTIPLEX COHERENT ANTI-STOCKES RAMAN SCATTERING MICROSCOPY (CARS-M). J. Jasensky,a A. Boughton,a A. Khmaladze,b J. E. Swain,c Z. Chen,a G. D. Smith.c aDepartment of Biophysics, University of Michigan, Ann Arbor, MI; bDepartment of Chemistry, University of Michigan, Ann Arbor, MI; cDepartment of OB/Gyn, University of Michigan, Ann Arbor, MI. OBJECTIVE: Cell survival post-cryopreservation is inversely related to intracellular lipids. No methods have been used to quantify oocyte/embryo lipids without sacrificing cells. We developed CARS-M to image chemical composition of cells non-invasively. Objectives were to evaluate CARS-M for intra-oocyte lipid quantification. DESIGN: Laboratory study. MATERIALS AND METHODS: Oocytes were germinal vesicle-intact (GVI) and used with IRB or UCUCA approval. Meiotically-incompetent (MeI) and -competent (MeC) GVI oocytes were collected from mouse preantral and antral follicles, respectively. Bovine, porcine and human GVI oocytes came from antral follicles. Oocytes were fixed/stained with Nile Red for control lipid quantification. A CARS-M was developed for non-invasive oocyte imaging. CARS is a vibrational spectroscopy that probes intrinsic chemical signatures such as CH2, a main component of lipids. Statistics were performed using t-test. RESULTS: CARS-M could be performed on living oocytes with a lipid signal of 2850cm1. Proof-of-concept studies on oocytes from species with known differences in lipids yielded similar results with fluorescent microscopy on fixed/stained oocytes and CARS-M on living oocytes. Lipid content was highest in porcine, moderate in bovine, and lowest in mouse and human oocytes. CARS studies on living mouse GVI oocytes demonstrated intracellular lipid content (% of cell volume) was less in MeI (1.1%
0.1) compared to MeC oocytes (2.2%
0.2; P<0.01). These differences were confirmed in fixed/stained oocytes (MeI: 1.1%
0.2 and MeI: 2.5%
0.2; P<0.01). CONCLUSION: CARS-M can be performed on living oocytes to obtain spectroscopic (chemical) and microscopic (morphological) information with a single device on single cells. CARS-M can quantify lipid differences between oocytes of different developmental competence and has clinical potential in non-invasive determination of lipid content and susceptibility to cryo-damage or determination of intracellular chemical signatures as biomarkers of oocyte/embryo quality.

O-267 Wednesday, October 24, 2012 11:45 AM LASER-ASSISTED HATCHING (LAH) OF CRYOPRESERVED EMBRYOS – THE SIGNIFICANCE OF HOLE SIZE. B. Link,a B. Wong,a J. Mayer,b M. Sullivan,a J. Fleetham,a C. Greene.a aRegional Fertility Program Inc., Calgary, AB, Canada; bEastern Virginia Medical School, Norfolk, VA. OBJECTIVE: To determine the impact of hole size in laser-assisted hatching of cryopreserved embryos. DESIGN: A prospective, randomized and controlled study comparing the percentage of fully hatched blastocysts by Day 7 (D7) of culture between five different LAH hole sizes (10, 15, 20, 30 and 40 mm) and control. The primary outcome of the study was the percentage of fully hatched blastocysts on D7. The percentage of blastocysts stuck at the inner cell mass (ICM) during hatching was also recorded to assess the potential increased risk of monozygotic twinning (MZT) with LAH. MATERIALS AND METHODS: 300 cleavage-stage cryopreserved embryos were randomized into five treatment groups and control. Based on data from a pilot study, this provides 95% power. LAH was performed using the Saturn Active Laser System on the treatment groups and the control underwent equivalent microscope exposure. Hatching progress was observed from Days 3-7. Data were analyzed using Chi-Square and t-Test as appropriate. RESULTS: All treatment groups except 10 mm showed significantly higher percentages of fully hatched blastocysts than the control on D7

S79

(P<0.05). Percentage of fully hatched blastocysts on D7 was not significantly different among these four treatment groups. The 10 and 15 mm groups both had significantly more blastocysts stuck at the ICM when attempting to hatch compared to the 30 and 40 mm groups (P<0.05).

CONCLUSION: A 30 mm hole is recommended when performing LAH in order to promote hatching and minimize the chance of blastocysts being stuck during the process. Fully hatched blastocysts may be associated with high implantation rates and when blastocysts become stuck at the ICM during hatching, MZT may occur after separation into two separate ICMs. Supported by: EMD Serono Canada.

DESIGN: Retrospective observational study. MATERIALS AND METHODS: Ovarian stimulation was performed by combination of GnRH agonist or antagonist and FSH/hMG. For ICSI, one hour after egg retrieval, the cumulus corona complex were stripped from the egg using enzyme and repetitive pipeting. For IVF, three hour after egg retrieval, oocyte-cumulus complex were inseminated by conventional insemination. Two pronuclei was checked after 1618 hours. From June 2010 to Nov 2011, 145 unfertilized mature oocytes following ICSI from 24 couples (Age¼37
3.9) and 138 unfertilized mature oocytes after IVF from 24 couples (Age¼35
5.0), were examined to see the number of spindle birefringence by the Polscope. RESULTS: The rate of one spindle in the ICSI group (42%, 61/145) was significantly lower than that of the IVF group (89.8%, 124/138) (P<0.05). After ICSI, two meiotic spindles were observed in (47.6%, 65/145) unfertilized oocytes, this is significantly higher than that (9.4%, 13/138) of the IVF group (P<0.05). The rate of unfertilized eggs which did not show any spindle position in ICSI and IVF group was 13% (19/145) and 1% (1/138), respectively. In addition, in the ICSI group there was 21.5% (14/65) of the unfertilized eggs shown two spindle birefringence and continued to cleavage the next day. But none developed to cleavage in the IVF group. CONCLUSION: These results suggest that failure of fertilization in IVF is most likely due to by the sperm unable to enter into the oocytes. In contrast, major reasons for fertilization failure after ICSI are due to incomplete oocyte activation even following sperm positioned.

O-268 Wednesday, October 24, 2012 12:00 PM

O-270 Wednesday, October 24, 2012 12:30 PM

INTRACYTOPLASMIC MORPHOLOGICALLY SELECTED SPERM INJECTION (IMSI) BENEFITS IN THE PRESENCE OF UNEXPLAINED INFERTILITY: A PROSPECTIVE RANDOMIZED STUDY. A. S. Setti,a D. P. A. F. Braga,a,b R. C. S. Figueira,b S. S. Colturato,b A. Iaconelli Jr.,a,b E. Borges Jr.a,b aSapientiae Institute - Educational and Research Center in Assisted Reproduction, Sao Paulo, SP, Brazil; bFertility - Assisted Fertilization Center, Sao Paulo, SP, Brazil.

A HIGHLY SENSITIVE ELECTROCHEMILUMINESCENCE IMMUNOASSAY FOR DETECTING EMBRYONIC HUMAN CHORIONIC GONADOTROPIN IN EMBRYO CULTURE MEDIA. J. Li,a X. Chen,a D. Jiang,b X. Liu,b Y. Liu,a G. Zhuang.a aHuman Reproductive Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China; bClinical Laboratory Department, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China.

OBJECTIVE: To evaluate the efficacy of the intracytoplasmic morphologically selected sperm injection (IMSI) in couples with unexplained infertility. DESIGN: Prospective randomized study. MATERIALS AND METHODS: This study included couples undergoing intracytoplasmic sperm injection (ICSI) as a result of unexplained infertility. A sample size calculation revealed that 36 cycles would be needed in each treatment arm to detect a 15% difference in implantation rate, with a significance level of 5% and power of 80%. Couples were randomly allocated to receive one of two sperm selection procedures (ICSI, n¼36; or IMSI, n¼36). The groups were compared with regard to cycles’ outcomes. RESULTS: There were no significant differences between ICSI and IMSI groups regarding the maternal age (32.3
2.8 vs 32.3
2.7; P¼1.000), number of follicles (15.8
5.5 vs 10.8
6.5; P¼0.463) and oocytes (12.1
8.8 vs 8.8
5.1; P¼0.780). Fertilization rate was significantly higher in IMSI group (66.0 vs 75.9%; P¼0.032). High-quality embryos rate (59.6 vs 53.5%; P¼0.412) and number of transferred embryos (2.7
0.6 vs 2.8
0.5; P¼0.546) were similar between ICSI and IMSI groups. IMSI cycles showed significantly higher implantation (19.4 vs 39.6%; P¼0.019) and pregnancy (33.3 vs 66.7%; P¼0.004) rates. The IMSI procedure positively influenced the implantation rate (RC: 35.39; R2: 7.7; P¼0.018) and was determinant to the increased odds of pregnancy (OR: 4.0; CI: 1.50 – 10.66; P¼0.004). CONCLUSION: This study demonstrates that, in couples with unexplained infertility, IMSI performance results in higher implantation and pregnancy rates as compared to conventional ICSI. It seems that the injection of a morphologically normal spermatozoon overcomes the hidden oocytes and/ or spermatozoa abnormalities that might affect couples with unexplained infertility, resulting in a 4-fold increase in pregnancy rate.

OBJECTIVE: To evaluate b-hCG in spent embryo culture media at day 3 or day 5 after fertilization by electrochemiluminescence immunoassay (ECLIA) and to explore the new assay as a biomarker for embryo assessment in clinical application. DESIGN: Experimental study. MATERIALS AND METHODS: A total of 118 spent culture media from day3 (41 samples) and day5 (77 samples, in which 41 were corresponding with day3 samples from the same embryos) were collected and quantified by ECLIA, and the blank controls and reliability test were performed as well. RESULTS: 1) 82.9% (34/41) in day 3 samples and 98.7% (76/77) in day 5 samples were detected b-hCG by ECLIA. 2) The concentration of b-hCG in culture media in day 5 group(n¼76) was significantly higher than it in day 3 group(n¼34) (1.57
0.71mIU/ml vs 0.51
0.23mIU/ml, P<0.001); the difference was also found in homologous samples (n¼33, 8 negative samples excluded) from the same embryo between day 5 group and day 3 group, (1.43
0.91mIU/ml vs 0.52
0.23mIU/ml, P<0.001). 3)There is no detectable amount of b-hCG in blank controls and the variation in homologous samples was less than 0.00007 mIU/ml. 4) The procedure was finished in twenty minutes for individual sample. The correlation coefficient between blastocyst grading and concentration of b-hCG was 0.525, P value<0.001. CONCLUSION: Based on our knowledge, the ECLIA was first reported as a perfect choice for detecting b-hCG in spent culture media for further embryo assessment, with the characteristics of the small sample requirement,speediness and high validity or reliability.This may have made itself a broad prospect in clinical application.

Hole Size (mm)

% Fully Hatched D7

% Stuck D7

18 (9/50) 34 (17/50) 52 (26/50)* 42 (21/50)* 50 (25/50)* 60 (30/50)*

8 (4/50) 14 (7/50) 12 (6/50) 2 (1/50) 0 (0/50)A 0 (0/50)A

Control 10 15 20 30 40

* Significant compared to Control. A Significant compared to the 10 and 15 mm groups.

O-269 Wednesday, October 24, 2012 12:15 PM SPINDLE EXAMINATION OF UNFERTILIZED EGGS BETWEEN ICSI AND IVF WITH POLSCOPE. J. H. Moon, J.-T. Chung, S. Henderson, S. G. Jin, A. Garcia, H. Holzer. Reproductive Center, Mcgill University Health Center, Montreal, QC, Canada. OBJECTIVE: Despite the entry of sperm by ICSI into mature eggs, failed fertilization still occurs. Cytological analyses of oocytes that fail to fertilize following ICSI have shown some sperm centrosomal dysfunction. In our study, we examined the appearance of spindles birefringence using Polscope on unfertilized eggs after failure of fertilization following ICSI, then compares the spindle pattern with those of IVF.

S80

ASRM Abstracts

The concentration of beta-hCG in different blastocyst score groups

blastocyst expansion and hatching status according to Gardner compact or morula 1 2 3 4 5 6

No. of samples

b-hCG (mIU/ml)

6 10 24 13 20 2 1

0.65
0.19 1.23
0.30 1.51
0.44 1.72
0.35 1.78
0.51 4.03
2.52 2.27

Vol. 98, No. 3, Supplement, September 2012