Saad Al-Hassan

Clinical application of multiple displacement amplification in preimplantation genetic diagnosis

Multiple displacement amplification (MDA) is a technique used in the amplification of very small amounts of DNA. MDA is reported to yield large quantities of high-quality DNA. The applicability of MDA to single cells was recently demonstrated as a potential technique for preimplantation genetic diagnosis (PGD). This paper shows the first clinical application of MDA in PGD. Two cycles of PGD were performed in two diseases, resulting in two pregnancies. All the diagnoses given on blastomeres were confirmed on the non-transferred whole embryos. The blastomere diagnosis was coupled with short tandem repeat (STR) analysis (16 loci) in all cycles. Allelic drop-out (ADO) assessment and amplification efficiency were evaluated on 40 single lymphocytes derived from parents of each disease. ADO and amplification failure were 10.3 and 2.2% for beta-thalassaemia and 17.9 and 2.2% for cystic fibrosis respectively. HLA matching for A, B and DR was performed successfully on single cell for the beta-thalassaemia family using similar methods to genomic DNA. The PGD protocol used in all diseases consists of MDA amplification, followed by a standard polymerase chain reaction protocol. Although HLA matching was not applied to embryos, its feasibility was shown on single cell DNA amplified by MDA. Altogether, these data show the simplicity and reliability of performing PGD in combination with HLA matching and STR analysis using MDA.

The correlation between endometrial thickness and outcome of in vitro fertilization and embryo transfer (IVF-ET) outcome

BackgroundTo evaluate the relationship between endometrial thickness on day of human chorionic gonadotrophin administration (hCG) and pregnancy outcome in a large number of consecutive in vitro fertilization and embryo transfer (IVF-ET) cycles.MethodsA retrospective cohort study including all patients who had IVF-ET from January 2003–December 2005 conducted at a tertiary center.ResultsA total of 2464 cycles were analysed. Pregnancy rate (PR) was 35.8%. PR increased linearly (r = 0.864) from 29.4% among patients with a lining of less than or equal to 6 mm, to 44.4% among patients with a lining of greater than or equal to 17 mm. ROC showed that endometrial thickness is not a good predictor of PR, so a definite cut-off value could not be established (AUC = 0.55).ConclusionThere is a positive linear relationship between the endometrial thickness measured on the day of hCG injection and PR, and is independent of other variables. Hence aiming for a thicker endometrium should be considered.

Y chromosome microdeletions in infertile men with idiopathic oligo- or azoospermia

About 30–40% of male infertility is due to unknown reasons. Genetic contributions to the disruption of spermatogenesis are suggested and amongst the genetic factors studied, Y chromosome microdeletions represent the most common one. Screening for microdeletions in AZFa, b and c region of Y chromosome showed a big variation among different studies. The purpose of this study was to investigate the prevalence of such deletions in Saudi men. A total of 257 patients with idiopathic oligo- or azoospermia were screened for Y chromosome microdeletions by 19 markers in AZF region. Ten (3.9%) patients had chromosomal rearrangements, six of them showed sex chromosome abnormalities and four patients had apparently balanced autosomal rearrengements. Eight of the remaining 247 patients (3.2%) with a normal karyotype and no known causes of impaired spermatogenesis had Y chromosome microdeletions. Among these, six patients had deletions in AZFc region, one case had a deletion in AZFb and another had both AZFa and AZFc deletions. In conclusion, our study shows that Y chromosome microdeletions are low in our population. We also report for the first time a case with unique point deletions of AZFa and AZFc regions. The lower frequency of deletions in our study suggest that other genetic, epigenetic, nutritional and local factors may be responsible for idiopathic oligo- or azoospermia in the Saudi population.

Intracytoplasmic sperm injection and conventional in vitro fertilization are complementary techniques in management of unexplained infertility.

AbstractPurpose: To evaluate the role of ICSI in unexplained infertility. Methods: In 125 cycles with six or more oocytes retrieved per cycle, sibling oocytes were randomly allocated to IVF or ICSI (group A). In 74 cycles with less than six oocytes retrieved per cycle, cycles were allocated to IVF or ICSI (group B). Results: In group A, ICSI fertilization rate of 61% per allocated oocyte was higher than IVF fertilization rate of 51.6% (P < 0.001). Complete fertilization failure occurred in 19.2 and 0.8% of cycles in IVF and ICSI, respectively (P < 0.001). In group B, fertilization rate in IVF cycles was 53.3% as compared to 60.7% per allocated oocyte in the ICSI cycles (P = 0.29). Complete fertilization failure was higher (P = 0.02) in conventional IVF (34.3%) than ICSI cycles (10.3%). Conclusions: Allocation of sibling oocytes to IVF and ICSI in the first cycle minimizes risk of fertilization failure. For patients with limited number of oocytes, ICSI technique is recommended.

Transcriptional profiling of granulosa cells from a patient with recurrent empty follicle syndrome

Empty follicle syndrome (EFS) is characterized by the absence of oocytes after apparently normal follicular development and the pathogenesis of this syndrome is not well characterized. The aim of this study was to analyse whole gene expression of granulosa cells (GC) from a patient with recurrent EFS by using Affymetrix GeneChip. A total of 160 genes were identified as being differentially expressed (by at least two-fold) between EFS GC and the control GC. Most of the differentially expressed genes were involved in cell growth and death. Among these were MAPK3, which plays an important role in the inhibition of apoptosis, was down-regulated 2.3-fold in EFS GC. Moreover, secretory phospholipase A2 and transforming growth factor receptor II, key regulators of cell death pathway, were down-regulated 3.54- and 2.82-fold respectively in EFS. Gene expression of granulosa cells from the EFS patient was significantly altered. The absence of the oocytes might be due to the increased apoptotic gene expression and the reduction of transcripts whose products are responsible for healthy follicular growth. Gene expression analyses might be a useful technique in identifying markers to follow a healthy follicular maturation and understanding the events that lead to EFS.

Advanced surgical sperm recovery is a viable option for intracytoplasmic sperm injection in patients with obstructive or nonobstructive azoospermia

OBJECTIVE To determine whether advanced sperm retrieval is appropriate in cases of obstructive and nonobstructive azoospermia. DESIGN Prospective controlled study. SETTING Tertiary care center. PATIENT(S) Men with obstructive and nonobstructive azoospermia, and their partners. INTERVENTION(S) Surgical sperm retrieval followed by intracytoplasmic sperm injection (ICSI) after 4 or 48 hours. MAIN OUTCOME MEASURE(S) Fertilization and pregnancy rates. RESULT(S) Advanced and fresh surgical sperm recoveries for ICSI were performed in 54 and 230 cycles, respectively. Patient demographics and cycle parameters were comparable. Two hundred forty-one (56.3%) of 428 injected eggs in the advanced retrieval group were fertilized, compared with 955 (56.6%) of 1,686 eggs in the fresh retrieved group (P=.94). There was no statistically significant difference in the pregnancy rates per ET between groups: 38.2% (18 of 47) in the advanced retrieval group and 39.9% (73 of 183) in the fresh sperm recovery group (P=.97). CONCLUSION(S) Testicular and epididymal sperm recovery can be safely performed 48 hours before ICSI. This facilitates planning, and, in cases of failure to retrieve sperm, hCG administration and ovum pick-up can be canceled, thereby reducing costs and eliminating the risk of ovarian hyperstimulation.

First successful application of preimplantation genetic diagnosis and haplotyping for congenital hyperinsulinism

Congenital hyperinsulinism is the most common cause of persistent hypoglycaemia in infancy. Early surgical intervention is usually required to prevent brain damage. The prevention of the transmission to the offspring is important in families carrying the mutated gene. Preimplantation genetic diagnosis (PGD) is an early genetic testing procedure for couples at risk of transmitting inherited diseases. A 36-year-old Saudi woman married to her first cousin with four affected children was referred for PGD. The hyperinsulinism disease was caused by a novel homozygous mutation in the KCNJ11 gene, an arginine 301 to proline (R301P) substitution.PGD was achieved by whole genome amplification followed by mutation detection combined with short tandem repeat identifier analysis in the first cycle and with haplotyping in the second cycle. The first and second cycles resulted in the births of healthy twin girls and a boy, respectively. As far as is known, this is the first application of PGD to hyperinsulinism. A feasible strategy including whole genome amplification followed by direct mutation detection combined with haplotyping is described.Utilizing haplotyping increases the efficiency of PGD diagnosis as well as confirming the genetic diagnosis. It reveals the parental origin of each inherited chromosome.

Low Plasma Levels of hCG After 10,000-IU hCG Injection Do Not Reduce the Number or Maturation of Oocytes Recovered in Patients Undergoing Assisted Reproduction

Purpose:Our purpose was to determine whether there is a correlation between human chorionic gonadotropin (hCG) blood levels and oocyte maturation.Methods:Three samples of blood were obtained at different times from hCG administration as follows: 12 hr, 36 hr, during oocyte recovery, and at 84 hr, when the patient comes for embryo transfer.Results:A total of 5036 oocytes was retrieved from 404 patients prospectively recruited between April 1996 and March 1997. The percentage of metaphase-II oocytes at different blood levels ranged from 84 to 88%. The general trend does not show any significant increase in percentage of metaphase-II oocytes in association with an increasing serum hCG concentration.Conclusions:The results of this study suggest that at 12, 36, and 84 hr after hCG administration, levels as low as 50, 45, and 9 IU/L of hCG, respectively, are equally potent as higher levels at initiating maximal oocyte maturity.

Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer.

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.