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PGD for aneuploidy: us IVF clinics' practice and policy
embryos comes from PGS of embryos from patients with impaired fertility. PGS for aneuploidy is not a common recommendation for the donor egg patient group. However, we and others have demonstrated preliminary evidence that aneuploidy rates in this patient group exceeds 40% of the embryos analyzed. Therefore, we determined aneuploidy rates in 46 couples undergoing donor egg IVF cycles. DESIGN: Retrospective study. MATERIALS AND METHODS: Forty-six couples underwent donor egg IVF-PGD due to poor outcomes from prior fertility therapy. Laser-assisted embryo biopsy was performed on day-3 and PGS was done on 601 cleaving embryos from 46 initiated cycles. The mean donor age was 26.5 years (range of 21 to 31). Multi-color fluorescence in situ hybridization (FISH) was used to determine aneuploidy for chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y. Hybridization, stringency washes and fluorescent microscopy was performed according to routine laboratory protocols. Clinical outcomes (aneuploidy, embryo transfer, clinical pregnancies and delivery rates) of these cycles were determined. Clinical pregnancy was defined by ultrasound identification of an intrauterine gestational sac and fetal heart beat. RESULTS: All 46 women had an embryo transfer. Five percent (30/601) of the embryos were not diagnosed due to poor blastomere quality. Fifty percent (301/601) of the embryos were abnormal for at least one of the 10 chromosomes tested. Over 70% of the genetically normal embryos developed into a blastocyst. The clinical pregnancy rate was 70% (32/46) per patient and per embryo transfer. There were no miscarriages, misdiagnosis, nor mosaic embryos. Sixty-three percent (20/32) of women delivered a healthy child while 37% (12/32) are ongoing pregnancies. CONCLUSIONS: This study from donor egg cycles provides insight into the presence of aneuploidy in a low risk population. Pregnancy rates were similar in these patients to those undergoing donor egg IVF without PGD. Supported by: None.
DESIGN: A retrospective data analyses. MATERIALS AND METHODS: Patients who underwent IVF or ICSI and also had PGD were included in the study. PGD was performed by FISH with probes for chromosomes 13, 18, 21, X and Y 3 days after egg retrieval. Additional chromosomes 8, 9, 14, 15, 16, 17, 20 and 22 were examined if indicated. Each embryo was scored by cell number and grade (A-D) on the day of PGD. RESULTS: 1. PGD was performed on 1119 embryos derived from IVF and 460 embryos derived from ICSI. Chromosomal abnormalities were 51.7% and 51.1% in embryos of IVF and ICSI groups, respectively. There were no differences in rates of polyploidy (7.0 vs. 5.9%), haploidy (1.7 vs. 1.3%), aneuploidy (25.0 vs. 17.4%), and complex chromosome abnormality (18.8 vs. 26.5%) between IVF and ICSI groups. 2. The rate of aneuploidy was not different among embryos with different numbers of blastomere (20– 30%). However, the rate of complex chromosome abnormality was significantly lower in embryos at 8 cell stage (9 vs. 5%) and higher in embryos with lower cell number (34 vs. 36%) for IVF and ICSI cycles (Fig 1). 3. Complex chromosome abnormality was also rare in embryos with good grade (8 vs. 4% for grade A) and common in embryos with poor grade (53 vs. 48%for grade D) for IVF and ICSI cycles (Fig 2). CONCLUSIONS: 1. The PGD results from both IVF and ICSI derived embryos revealed a correlation between the incidence of complex chromosome abnormality and cell number of embryos. 2. Slow growing embryos are associated with more complex chromosome abnormality. 3. Embryo grade is the most important predictor for complex chromosome abnormality. 4. ICSI, performed for male infertility, doesn’t change chromosomal pattern revealed by PGD.
P-400 COMPLEX CHROMOSOME ABNORMALITY IN PATIENTS WITH IN VITRO FERTILIZATION (IVF) AND INTRACYTOPLASMIC SPERM INJECTION (ICSI). S. P. Weng, T. C. J. Wu, S. P. Kang, M. W. Surrey, H. C. Danzer, D. L. Hill. Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, David Geffen School of Medicine at UCLA, Los Angeles, CA; ART Reproductive Center, Beverly Hills, CA. OBJECTIVE: To evaluate the incidence of complex chromosome abnormality in embryos derived from IVF and ICSI and its relationship to embryo stages and grading.
Figure 2.
Supported by: None.
P-401 PGD FOR ANEUPLOIDY: US IVF CLINICS’ PRACTICE AND POLICY. S. Baruch, D. Kaufman, K. L. Hudson. Genetics and Public Policy Center, Johns Hopkins University, Washington, DC.
Figure 1.
FERTILITY & STERILITYÒ
OBJECTIVE: To understand the use of Preimplantation Genetic Diagnosis (PGD) for aneuploidy screening by United States ART providers, and the attitudes of clinic directors towards this use of PGD. DESIGN: An on-line survey of directors of United States IVF clinics, or their designees.
S239
MATERIALS AND METHODS: In 2006 the Genetics and Public Policy Center conducted a survey of all known United States ART clinics (1) to better understand current practices of ART clinics offering PGD to patients and (2) to explore the attitudes and opinions of clinic directors towards PGD, including its uses for aneuploidy. Contact information for ART clinic directors came from the Society for Assisted Reproductive Technology (SART) and publicly available information from the Centers for Disease Control. RESULTS: Our survey of 415 ART clinics had a valid response rate of 45% or 186 clinics. We found that 93% of IVF clinics that perform PGD (68% of all responding IVF clinics) have provided PGD for aneuploidy. These clinics reported performing a total of 2,197 PGD aneuploidy cycles in 2005. Extrapolating from our respondents to all IVF clinics we estimate that 5,000 cycles of PGD were performed to detect aneuploidy in U.S. IVF clinics in 2005. Most (89%) directors of IVF clinics performing PGD for aneuploidy do not believe that PGD aneuploidy screening should be offered to all or most IVF patients. However, 52% believe that in the future PGD will be offered to all or most couples undergoing IVF. More clinics directors providing PGD aneuploidy screening believe it is a valid tool when a patient has had repeated miscarriages (93%) than when a patient has had repeated IVF failure (79%) or when a patient is of advanced maternal age (71%). The overwhelming majority (85%) of directors performing PGD aneuploidy screening believe more data are needed to determine whether and to whom it should be offered. Nearly half (47%) of the directors who offer PGD aneuploidy screening agreed with the statement ‘‘the push to offer PGD for aneuploidy screening is more about market pressure than medical evidence’’. CONCLUSIONS: PGD for aneuploidy is by far the most common indication for PGD in the United States. However, views about which patient groups it is effective for vary greatly. Additional research is needed to determine when PGD for aneuploidy is appropriate. There may be a role for professional guidelines in this area. Supported by: The Pew Charitable Trusts.
P-402
GENETIC COUNSELING P-403 INTERCHROMOSOMAL EFFECTS IN SPERM OF THREE HETEROZYGOUS CARRIERS OF STRUCTURAL CHROMOSOMAL REARRANGEMENTS. G. Kirkpatrick, E. Chan Wang, K. Ferguson, S. Ma. Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada. OBJECTIVE: It is well known that men with balanced chromosomal rearrangement are at risk of producing chromosomally unbalanced gametes. However, it is not clear if the meiotic behavior of a balanced chromosomal rearrangement could affect the segregation of chromosomes not involved in the rearrangement (interchromosomal effect), leading to aneuploidy in the gametes. In this study, we investigated the existence of an interchromosomal effect in three heterozygous carriers of structural chromosomal rearrangements. DESIGN: Prospective analysis of the chromosomal complements of spermatozoa by fluorescent in situ hybridization (FISH). MATERIALS AND METHODS: All three carriers had abnormal sperm parameters, and history of infertility at least two years. Semen samples were collected from a carrier of a paracentric inversion: inv (5) (q22.1;q23.2); a reciprocal translocation: t (9;22) (p13.1;q13.2); and a Robertsonian translocation: t (13;21) (q10;q10), as well as five normal men. Spermatozoa were prepared for dual FISH for chromosomes 13 and 21, and triple FISH for chromosomes 18, X and Y. 10,000 sperm were scored per patient for each probe set. The Chi-square test was used to compare rates of disomy for each chromosome between the patients and the control men. P<0.05 was considered significant. RESULTS: A total of 152,436 spermatozoa were scored (101,492 controls and 50,944 for patients). Evidence of an interchromosomal effect was observed in the inv(5) carrier, who displayed significantly increased disomy 13, 21, 18, XX and YY (Table 1). The t (9;22) reciprocal translocation carrier showed increased disomy 21, but aneuploidies for all other chromosomes studied were not increased. There was no evidence of an interchromosomal effect in the t (13;21) Robertsonian translocation carrier.
PRE-IMPLANTATION GENETIC DIAGNOSIS IN A CASE OF RECURRENT TRISOMY 21 OFFSPRING. S. B. A. Hudson, D. L. Walker, J. R. Fredrickson, D. E. Morbeck, C. C. Coddington, R. P. Gada. Obstetrics and Gynecology, Mayo Clinic, Rochester, MN. OBJECTIVE: To describe a unique case of recurrent aneuploidy and the utilization of pre-implantation genetic diagnosis (PGD). DESIGN: Case report. MATERIALS AND METHODS: PGD performed on cryopreserved embryos of a couple with two prior Trisomy 21 children. RESULTS: A 31 year old G0 Caucasian female initially presented to a Midwest reproductive endocrinology and infertility clinic after 2 years of infertility. Initial evaluation was negative including family history except minimal endometriosis on laparoscopy. Patient underwent clomiphene citrate ovulation induction then gonadotropin superovulation with intrauterine insemination without success. At age 34, the patient decided to proceed with in vitro fertilization (IVF). The patient became pregnant with a Trisomy 21 infant utilizing a fresh IVF cycle and delivered via low transverse Cesarean section for breech presentation. Genetic analysis of both parents revealed normal karyotypes. The patient then spontaneously conceived another infant with Trisomy 21 at age 35 and delivered via low forceps for placental abruption and fetal heart rate abnormalities. The patient and her husband then inquired about evaluating the three remaining frozen embryos with PGD. The patient was then counseled of the risks and benefits of PGD including the possibility of false positive and negative results. The three embryos were evaluated utilizing PGD and florescence in situ hybridization. One embryo noted complex aneuploidy. Another embryo was indeterminate for monosomy 16 vs. overlapping signals of two chromosome 16 markers. This embryo was transferred along with the third embryo which appeared chromosomally normal. The patient delivered a chromosomally normal infant at 36 weeks gestation via low transverse Cesarean section for breech presentation. CONCLUSIONS: Multiple modalities are currently available for prenatal evaluation of Down’s syndrome including quadruple screen, chorionic villus sampling, nuchal translucency, anatomic ultrasound evaluation and amniocentesis. However, each of these is performed after conception and leaves the prospective parents with the decision of continuing vs. terminating the pregnancy. In select cases in patients with an increased risk of aneuploidy, PGD may prove useful as a diagnostic tool to help ensure a euploid pregnancy. Supported by: None.
S240
Abstracts
TABLE 1. Incidence of sperm disomy for chromosomes 13, 18, 21 X an patients and controls
inv 5(q22.1;q23.2) t(9;22)(p13.1;q13.2) t(13;21)(q10;q10) Controls
a
No. sperm
Disomy 13 (%)
Disomy 21 (%)
No. sperm
Disomy 38 (%)
10,140 10,033 n/a 50,752
47a (0.46) 16 (0.16) n/a 50 (0.10)
74a (0.73) 53a (0.53) n/a 74 (0.15)
10,296 10,303 10,172 50,740
27a (0.26) 5 (0.05) 3 (0.03) 35 (0.07)
Sex chromosomes disomy (%) XX 48a (0.47) 2 (0.02) 0 (0) 42 (0.08)
YY 58a (0.56) 10 (0.10) 4 (0.04) 55 (0.11)
XY 14 (0.14) 13 (0.13) 11 (0.11) 99 (0.20)
P<0.01.
CONCLUSIONS: The existence of an interchromosomal effect in carriers of structural rearrangements is contentious, with some studies observing such an effect, and some not. In this study the inversion carrier showed evidence of an interchromosomal effect involving all chromosomes studied, while the t (9;22) carrier showed only an increased aneuploidy for chromosome 21. There was no evidence of an interchromosomal effect in the t (13;21) Robertsonian translocation carrier. Thus, the type of structural abnormality and the chromosomes and breakpoints involved may determine the presence and magnitude of an interchromosomal effect. Supported by: This study was supported by a grant from Canadian Institutes of Health Research (CIHR).
P-404 REPRODUCTIVE GENETIC COUNSELING IN PATIENTS WITH COMPLEX CHROMOSOMAL REARRANGEMENT. N. Takeshita, Y. Katagiri, Y. Shibui, M. Kitamura, Y. Fukuda, M. Morita. Obstetrics and Gynecology, Center for Reproductive Medicine and Infertility, Toho Medical Center, Omori Hospital, Toho University, Tokyo, Ohmori-Nishi, Ohta-Ku, Japan. OBJECTIVE: In this time, we conducted reproductive genetic counseling for a couple with a complex chromosomal rearrangement (CCR) that had five cleavage points involving four chromosomes 1, 6, 9 and 14. Here we present this case with some discussion regarding key points and matters to be considered for such counseling.
Vol. 88, Suppl 1, September 2007
DESIGN: A retrospective data analyses. MATERIALS AND METHODS: Patients who underwent IVF or ICSI and also had PGD were included in the study. PGD was performed by FISH with probes for chromosomes 13, 18, 21, X and Y 3 days after egg retrieval. Additional chromosomes 8, 9, 14, 15, 16, 17, 20 and 22 were examined if indicated. Each embryo was scored by cell number and grade (A-D) on the day of PGD. RESULTS: 1. PGD was performed on 1119 embryos derived from IVF and 460 embryos derived from ICSI. Chromosomal abnormalities were 51.7% and 51.1% in embryos of IVF and ICSI groups, respectively. There were no differences in rates of polyploidy (7.0 vs. 5.9%), haploidy (1.7 vs. 1.3%), aneuploidy (25.0 vs. 17.4%), and complex chromosome abnormality (18.8 vs. 26.5%) between IVF and ICSI groups. 2. The rate of aneuploidy was not different among embryos with different numbers of blastomere (20– 30%). However, the rate of complex chromosome abnormality was significantly lower in embryos at 8 cell stage (9 vs. 5%) and higher in embryos with lower cell number (34 vs. 36%) for IVF and ICSI cycles (Fig 1). 3. Complex chromosome abnormality was also rare in embryos with good grade (8 vs. 4% for grade A) and common in embryos with poor grade (53 vs. 48%for grade D) for IVF and ICSI cycles (Fig 2). CONCLUSIONS: 1. The PGD results from both IVF and ICSI derived embryos revealed a correlation between the incidence of complex chromosome abnormality and cell number of embryos. 2. Slow growing embryos are associated with more complex chromosome abnormality. 3. Embryo grade is the most important predictor for complex chromosome abnormality. 4. ICSI, performed for male infertility, doesn’t change chromosomal pattern revealed by PGD.
P-400 COMPLEX CHROMOSOME ABNORMALITY IN PATIENTS WITH IN VITRO FERTILIZATION (IVF) AND INTRACYTOPLASMIC SPERM INJECTION (ICSI). S. P. Weng, T. C. J. Wu, S. P. Kang, M. W. Surrey, H. C. Danzer, D. L. Hill. Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, David Geffen School of Medicine at UCLA, Los Angeles, CA; ART Reproductive Center, Beverly Hills, CA. OBJECTIVE: To evaluate the incidence of complex chromosome abnormality in embryos derived from IVF and ICSI and its relationship to embryo stages and grading.
Figure 2.
Supported by: None.
P-401 PGD FOR ANEUPLOIDY: US IVF CLINICS’ PRACTICE AND POLICY. S. Baruch, D. Kaufman, K. L. Hudson. Genetics and Public Policy Center, Johns Hopkins University, Washington, DC.
Figure 1.
FERTILITY & STERILITYÒ
OBJECTIVE: To understand the use of Preimplantation Genetic Diagnosis (PGD) for aneuploidy screening by United States ART providers, and the attitudes of clinic directors towards this use of PGD. DESIGN: An on-line survey of directors of United States IVF clinics, or their designees.
S239
MATERIALS AND METHODS: In 2006 the Genetics and Public Policy Center conducted a survey of all known United States ART clinics (1) to better understand current practices of ART clinics offering PGD to patients and (2) to explore the attitudes and opinions of clinic directors towards PGD, including its uses for aneuploidy. Contact information for ART clinic directors came from the Society for Assisted Reproductive Technology (SART) and publicly available information from the Centers for Disease Control. RESULTS: Our survey of 415 ART clinics had a valid response rate of 45% or 186 clinics. We found that 93% of IVF clinics that perform PGD (68% of all responding IVF clinics) have provided PGD for aneuploidy. These clinics reported performing a total of 2,197 PGD aneuploidy cycles in 2005. Extrapolating from our respondents to all IVF clinics we estimate that 5,000 cycles of PGD were performed to detect aneuploidy in U.S. IVF clinics in 2005. Most (89%) directors of IVF clinics performing PGD for aneuploidy do not believe that PGD aneuploidy screening should be offered to all or most IVF patients. However, 52% believe that in the future PGD will be offered to all or most couples undergoing IVF. More clinics directors providing PGD aneuploidy screening believe it is a valid tool when a patient has had repeated miscarriages (93%) than when a patient has had repeated IVF failure (79%) or when a patient is of advanced maternal age (71%). The overwhelming majority (85%) of directors performing PGD aneuploidy screening believe more data are needed to determine whether and to whom it should be offered. Nearly half (47%) of the directors who offer PGD aneuploidy screening agreed with the statement ‘‘the push to offer PGD for aneuploidy screening is more about market pressure than medical evidence’’. CONCLUSIONS: PGD for aneuploidy is by far the most common indication for PGD in the United States. However, views about which patient groups it is effective for vary greatly. Additional research is needed to determine when PGD for aneuploidy is appropriate. There may be a role for professional guidelines in this area. Supported by: The Pew Charitable Trusts.
P-402
GENETIC COUNSELING P-403 INTERCHROMOSOMAL EFFECTS IN SPERM OF THREE HETEROZYGOUS CARRIERS OF STRUCTURAL CHROMOSOMAL REARRANGEMENTS. G. Kirkpatrick, E. Chan Wang, K. Ferguson, S. Ma. Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada. OBJECTIVE: It is well known that men with balanced chromosomal rearrangement are at risk of producing chromosomally unbalanced gametes. However, it is not clear if the meiotic behavior of a balanced chromosomal rearrangement could affect the segregation of chromosomes not involved in the rearrangement (interchromosomal effect), leading to aneuploidy in the gametes. In this study, we investigated the existence of an interchromosomal effect in three heterozygous carriers of structural chromosomal rearrangements. DESIGN: Prospective analysis of the chromosomal complements of spermatozoa by fluorescent in situ hybridization (FISH). MATERIALS AND METHODS: All three carriers had abnormal sperm parameters, and history of infertility at least two years. Semen samples were collected from a carrier of a paracentric inversion: inv (5) (q22.1;q23.2); a reciprocal translocation: t (9;22) (p13.1;q13.2); and a Robertsonian translocation: t (13;21) (q10;q10), as well as five normal men. Spermatozoa were prepared for dual FISH for chromosomes 13 and 21, and triple FISH for chromosomes 18, X and Y. 10,000 sperm were scored per patient for each probe set. The Chi-square test was used to compare rates of disomy for each chromosome between the patients and the control men. P<0.05 was considered significant. RESULTS: A total of 152,436 spermatozoa were scored (101,492 controls and 50,944 for patients). Evidence of an interchromosomal effect was observed in the inv(5) carrier, who displayed significantly increased disomy 13, 21, 18, XX and YY (Table 1). The t (9;22) reciprocal translocation carrier showed increased disomy 21, but aneuploidies for all other chromosomes studied were not increased. There was no evidence of an interchromosomal effect in the t (13;21) Robertsonian translocation carrier.
PRE-IMPLANTATION GENETIC DIAGNOSIS IN A CASE OF RECURRENT TRISOMY 21 OFFSPRING. S. B. A. Hudson, D. L. Walker, J. R. Fredrickson, D. E. Morbeck, C. C. Coddington, R. P. Gada. Obstetrics and Gynecology, Mayo Clinic, Rochester, MN. OBJECTIVE: To describe a unique case of recurrent aneuploidy and the utilization of pre-implantation genetic diagnosis (PGD). DESIGN: Case report. MATERIALS AND METHODS: PGD performed on cryopreserved embryos of a couple with two prior Trisomy 21 children. RESULTS: A 31 year old G0 Caucasian female initially presented to a Midwest reproductive endocrinology and infertility clinic after 2 years of infertility. Initial evaluation was negative including family history except minimal endometriosis on laparoscopy. Patient underwent clomiphene citrate ovulation induction then gonadotropin superovulation with intrauterine insemination without success. At age 34, the patient decided to proceed with in vitro fertilization (IVF). The patient became pregnant with a Trisomy 21 infant utilizing a fresh IVF cycle and delivered via low transverse Cesarean section for breech presentation. Genetic analysis of both parents revealed normal karyotypes. The patient then spontaneously conceived another infant with Trisomy 21 at age 35 and delivered via low forceps for placental abruption and fetal heart rate abnormalities. The patient and her husband then inquired about evaluating the three remaining frozen embryos with PGD. The patient was then counseled of the risks and benefits of PGD including the possibility of false positive and negative results. The three embryos were evaluated utilizing PGD and florescence in situ hybridization. One embryo noted complex aneuploidy. Another embryo was indeterminate for monosomy 16 vs. overlapping signals of two chromosome 16 markers. This embryo was transferred along with the third embryo which appeared chromosomally normal. The patient delivered a chromosomally normal infant at 36 weeks gestation via low transverse Cesarean section for breech presentation. CONCLUSIONS: Multiple modalities are currently available for prenatal evaluation of Down’s syndrome including quadruple screen, chorionic villus sampling, nuchal translucency, anatomic ultrasound evaluation and amniocentesis. However, each of these is performed after conception and leaves the prospective parents with the decision of continuing vs. terminating the pregnancy. In select cases in patients with an increased risk of aneuploidy, PGD may prove useful as a diagnostic tool to help ensure a euploid pregnancy. Supported by: None.
S240
Abstracts
TABLE 1. Incidence of sperm disomy for chromosomes 13, 18, 21 X an patients and controls
inv 5(q22.1;q23.2) t(9;22)(p13.1;q13.2) t(13;21)(q10;q10) Controls
a
No. sperm
Disomy 13 (%)
Disomy 21 (%)
No. sperm
Disomy 38 (%)
10,140 10,033 n/a 50,752
47a (0.46) 16 (0.16) n/a 50 (0.10)
74a (0.73) 53a (0.53) n/a 74 (0.15)
10,296 10,303 10,172 50,740
27a (0.26) 5 (0.05) 3 (0.03) 35 (0.07)
Sex chromosomes disomy (%) XX 48a (0.47) 2 (0.02) 0 (0) 42 (0.08)
YY 58a (0.56) 10 (0.10) 4 (0.04) 55 (0.11)
XY 14 (0.14) 13 (0.13) 11 (0.11) 99 (0.20)
P<0.01.
CONCLUSIONS: The existence of an interchromosomal effect in carriers of structural rearrangements is contentious, with some studies observing such an effect, and some not. In this study the inversion carrier showed evidence of an interchromosomal effect involving all chromosomes studied, while the t (9;22) carrier showed only an increased aneuploidy for chromosome 21. There was no evidence of an interchromosomal effect in the t (13;21) Robertsonian translocation carrier. Thus, the type of structural abnormality and the chromosomes and breakpoints involved may determine the presence and magnitude of an interchromosomal effect. Supported by: This study was supported by a grant from Canadian Institutes of Health Research (CIHR).
P-404 REPRODUCTIVE GENETIC COUNSELING IN PATIENTS WITH COMPLEX CHROMOSOMAL REARRANGEMENT. N. Takeshita, Y. Katagiri, Y. Shibui, M. Kitamura, Y. Fukuda, M. Morita. Obstetrics and Gynecology, Center for Reproductive Medicine and Infertility, Toho Medical Center, Omori Hospital, Toho University, Tokyo, Ohmori-Nishi, Ohta-Ku, Japan. OBJECTIVE: In this time, we conducted reproductive genetic counseling for a couple with a complex chromosomal rearrangement (CCR) that had five cleavage points involving four chromosomes 1, 6, 9 and 14. Here we present this case with some discussion regarding key points and matters to be considered for such counseling.
Vol. 88, Suppl 1, September 2007