Levels of DNA adducts in the blood and follicular fluid of women undergoing in vitro fertilization treatment and its correlation with the pregnancy outcome.

This study is designed to investigate the impact of DNA damage on pregnancy and fertilization rate outcome in a sub-sample of women undergoing IVF treatment. Blood and follicular fluid samples (n = 60) were analyzed for DNA adducts. While no BPDE-DNA adducts were detected, other unknown lipophilic adducts were seen in blood and follicular fluid. Women who failed to achieve pregnancy had higher DNA adducts in follicular fluid than those who succeeded (p < 0.05). Follicular fluid cotinine levels were associated with DNA adduct levels in blood and follicular fluid (p < 0.05). Evaluation of DNA damage resulting from oxidative stress could have a role in predicting IVF success rate.

Outcome of in-vitro fertilization treatment and DDT levels in serum and follicular fluid.

BACKGROUND: Although p,p'-dichlorodiphenyltrichloroethane (DDT) is banned for agricultural purpose in Saudi Arabia, it is occasionally used to control vector-borne diseases in certain regions of the country. MATERIAL/METHODS: A case-control study was designed to investigate the possible effects of DDT and its metabolites on pregnancy and fertilization rate outcome. The study population was composed of 619 Saudi women (age 19-50 years) who sought in-vitro fertilization (IVF) treatment between 2002 and 2003. RESULTS: p,p'-DDE, the main metabolite of DDT, was the most frequently detected residue in serum or follicular fluid, with mean values of 1.646 microg/L and 0.407 microg/L, respectively. After controlling for many potential confounding variables, multiple logistic regression analysis revealed no association between pregnancy outcome or fertilization rate and p,p'-DDE levels in serum or follicular fluid. CONCLUSIONS: The inability to identify an effect may be related to the comparatively low concentrations of DDE in our population. But because p,p'-DDE was detected in the serum of 77.7% our participants, it should be considered as a matter of public heath concern. Currently there is no active source of DDT in our region; therefore, further studies are needed to identify sources in order to develop preventive measures because we can not exclude its potential reproductive toxicity.

Exposure to heavy metals (lead, cadmium and mercury) and its effect on the outcome of in-vitro fertilization treatment.

We investigated the effect of lead, cadmium and mercury exposure on pregnancy and fertilization rate outcome among 619 Saudi women (age 19-50 years) who sought in-vitro fertilization (IVF) treatment between 2002 and 2003. The concentrations of lead, cadmium and mercury were measured in both blood and follicular fluids. At levels well below the current US occupational exposure limit guidelines (40microg/dL) and even less than the current Centers for Disease Control and Prevention level of concern for preventing lead poisoning in children (10microg/dL), blood lead level was negatively associated with fertilization outcome in both adjusted and unadjusted logistic regression models. We found that among various demographic, socioeconomic and environmental factors, fish consumption was positively associated with blood lead levels. These results support the hypothesis that a raised blood lead level affects infertility and intervention to reduce the lead exposure might be needed for women of reproductive age. The present results also revealed unexpected finding - the positive relationship between follicular cadmium levels and fertilization outcome, which points to the necessity for further investigation. Though adverse effect of mercury on pregnancy outcome or fertilization rate was not evident in this study, mercury5.8microg/L (EPA safety limit) was found in the blood and follicular fluid of 18.7% and 8.3% of the women, respectively. Concerns about its possible adverse effects on the physiology of reproduction or fetal development cannot be ruled out. It should be noted that skin-lightening creams and dental amalgam were important contributors to mercury exposure. Such finding is alarming and priority for further studies are, urgently, needed.

Preimplantation genetic diagnosis for Niemann-Pick disease type B.

BACKGROUND: Acid sphingomyelinase (ASM) deficient Niemann-Pick disease (NPD) is an autosomal recessive disorder caused by mutations in the ASM gene (SMPD1). More than 70 different mutations have been reported in this gene. NPD type B is the most common type in Saudi Arabia with a frequency of 1:40 000 to 1:100 000. The phenotype of Saudi Type B patients is more severe than patients reported from the West. Two mutations specific to Saudi patients have been inherited in the SMPD1 gene. Given the difficult management of the disease, we opted for a preventive approach to the suffering families by screening the whole SMPD1 gene for mutations followed by Preimplantation Genetic Diagnosis (PGD). METHODS: The family suffering from NPD-B underwent mutation screening for the entire SMPD1 gene followed by PGD using nested PCR and sequencing. RESULTS: A novel mutation in a family suffering from the same severe NPD-B phenotype is described in this report (W533R). After PGD, a singleton pregnancy ensued after transfer of one heterozygous and one normal embryo. Postnatal DNA testing of the newborn showed a normal homozygous genotype. CONCLUSIONS: This report reveals a new SMPD1 mutation responsible for similar Saudi severe phenotype, and the prevention of this disorder by PGD.

Pregnancy after preimplantation genetic diagnosis for Sanjad-Sakati syndrome.

Sanjad-Sakati syndrome (SSS) is an autosomal recessive disorder characterized by congenital hypoparathyroidism, growth and mental retardation. In Saudi Arabia, the disease is caused by a deletion of 12 bp (155-166nt) in the tubulin-specific chaperone E gene. In a family with two affected siblings with SSS, preimplantation genetic diagnosis (PGD) was performed. Fluorescent PCR (F-PCR) was utilized to check the heterozygosity and the homozygosity status of the parents and the affected children, respectively. F-PCR was then optimized for single-cell analysis by using peripheral blood lymphocytes. The patient underwent a cycle with intra-cytoplasmic sperm injection. A total of 11 embryos were obtained and biopsied. There were five heterozygous, three homozygous affected and three normal embryos. One heterozygous and one normal embryo were transferred because of their very good quality (morula). A singleton pregnancy was obtained, and amniosynthesis confirmed the presence of the heterozygous fetus. These results show for the first time, the feasibility of PGD for SSS.

Zygote transfer on day 1 versus cleavage stage embryo transfer on day 3: a prospective randomized trial.

BACKGROUND: It has been reported that pronuclear morphology is related to embryo quality and viability, and that zygote stage embryos might establish pregnancies after being transferred to the uterus. The objective of this study was to investigate whether transferring zygotes on day 1 would result in similar pregnancy rates compared to transferring cleavage stage embryos on day 3 in a prospective randomized trial. METHODS: Patients undergoing IVF/ICSI treatments were randomized to either day 1 or day 3 transfers by envelope withdrawal technique. Zygotes were classified as 'pattern 0' and 'non-pattern 0' according to the size and alignment of pronuclei, the number and distribution of nucleoli. The two best zygotes or embryos were transferred on day 1 or day 3 respectively. The primary outcome measure was pregnancy rate. RESULTS: Pregnancy rates were higher in day 3 group (55/131, 42%) when compared to day 1 (34/123, 28%, P = 0.024). Similarly, implantation rates were higher in day 3 group (P = 0.03). There were more cycles with cryopreservation in the day 1 group (P < 0.001). Embryo quality on day 3 was similar between pattern 0 and non-pattern 0 zygotes. CONCLUSIONS: Day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers. The present pronuclei scoring cannot reliably select zygotes for transfer on day 1.

Is recombinant follicle-stimulating hormone more effective in IVF poor responders than human menopausal gonadotrophins?

BACKGROUND: The objective of this study was to determine if the recombinant human follicle-stimulating hormone (rFSH) is more effective than human menopausal gonadotrophin (hMG) in IVF poor responders. MATERIAL/METHODS: A prospective comparative study over a 2-year period. The setting was the Tertiary IVF Center, King Faisal Specialist Hospital and Research Center. A total of 150 patients were selected from 277 patients with poor response in previous hMG-stimulated cycles who were willing to undergo another cycle of treatment. Seventy-five patients stimulated with rFSH were compared with 75 control subjects (matched for age, early follicular phase FSH, and body mass index) stimulated with urinary hMG. The number of follicles, number of oocytes retrieved, cycle cancellations, and pregnancy rates were the main outcome measures. RESULTS: There were no statistical differences in numbers of follicles (6.2 versus 5.7; p=0.97), oocytes recovered (4 versus 3.3; p=0.15), cycle characteristics, or pregnancy rates between hMG- and rFSH-stimulated cycles (p=0.32). CONCLUSIONS: Recombinant follicle-stimulating hormone (rFSH) has no advantage over urinary human menopausal gonadotrophin (hMG) on ovarian performance or the outcome of IVF-ET in poor responders' IVF cycles.

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

PURPOSE: 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.

Nucleolar precursor body distribution in pronuclei is correlated to chromosomal abnormalities in embryos.

In-vitro generated human embryos have low implantation rates and high chromosomal abnormalities. Embryos are mostly selected on the basis of microscopic morphological examination. The relationship between pronuclear morphology and chromosomal abnormalities was investigated in this study. Zygotes were scored according to pronuclear morphology on day 1. Excess embryos that were not transferred or cryopreserved on day 3 were fixed. Chromosomes 13, 18, 21, X and Y were analysed by fluorescence in-situ hybridization (FISH). A total of 125 embryos were analysed; 58 (46%) were abnormal, 32 (26%) were mosaic and 35 (28%) were normal. Results were analysed according to different pronuclear morphology. Zygotes with polarized pattern had a significantly lower incidence of chromosome abnormality than those with a non-polarized pattern. The presence of cytoplasmic halo, the size of each pronucleus and the number of nucleolar precursor body had no significant effect on chromosomal abnormalities. In conclusion, embryos generated from zygotes with polarized pattern have fewer chromosomal abnormalities compared with other patterns. A simple microscopic examination during fertilization confirmation would be useful to select embryos with fewer chromosomal abnormalities, preferably in combination with other observations shown to correlate with chromosomal abnormalities.

Pregnancy after preimplantation genetic diagnosis for Ataxia Telangiectasia.

Ataxia Telangiectasia (AT) is an autosomal recessive disorder with an incidence estimated at 1 in 40 000 to 1 in 100 000 live births. More than 100 different somatic and germ-line mutations have been identified in the AT gene, the majority of which cause premature protein truncation. The immense size of the AT gene (66 exons) complicates the detection of mutations. A Saudi family with three affected children suffering from AT consulted our IVF centre for preimplantation genetic diagnosis (PGD). Despite advanced maternal age and unknown mutation, the family was screened for AT mutations. A large deletion in the gene was found to be responsible for the phenotype of AT. The mutation detection permitted us to perform PGD on AT for the first time. Single cell PCR consisted of amplifying one of the deleted exons, exon 19. Homozygous affected embryos show an absence of the exon, while in heterozygous or normal embryos the exon is amplified successfully. After ICSI, three embryos were suitable for embryo biopsy. After biopsy only one embryo showed exon amplification and was transferred. A singleton pregnancy ensued and prenatal diagnosis confirmed the presence of exon 19. This report demonstrates that PGD is feasible despite advanced maternal age and poor response to follicle stimulation.

Flow cytometric ploidy analysis of testicular biopsies from sperm-negative wet preparations.

BACKGROUND: The use of testicular sperm in assisted reproduction depends on the availability of sperm in wet preparations. It is not always possible to recover sperm from the testis, even with previous sperm-positive histopathological findings. The purpose of this study was to evaluate the sperm-negative wet preparation search results with flow cytometric ploidy analysis and histopathological examination. METHODS: Two pieces of testicular tissue were obtained from azoospermic patients to investigate the spermatogenic status of the testis, and to determine the presence of sperm through a wet preparation. The testicular tissue was shredded and then vortexed; the cellular suspension was then processed for a wet preparation sperm search, while the residual tissue was exposed to enzymatic digestion for flow cytometric ploidy analysis. RESULTS: A total of 38 patients had sperm-negative wet preparation results. Of those, six (16%) were shown to have haploid cells after flow cytometric analysis. Histopathological examination showed three samples with maturation arrest at the spermatid stage, and the other three at the spermatocyte stage. CONCLUSIONS: Flow cytometric ploidy analysis can be used to verify the results of a wet preparation sperm search when no sperm were detected. Flow cytometric ploidy analysis can also reveal the presence of spermatids when no sperm are available.

Value of flow cytometric assay for the detection of antisperm antibodies in women with a history of recurrent abortion.

PROBLEM: To verify the proposed relationship between recurrent spontaneous abortions and the presence of maternal antisperm antibodies (ASA) in women as detected by a sensitive and reliable method. METHOD OF STUDY: The presence of maternal antipaternal immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies were determined against three different paternal antigens comprising T, B lymphocytes and semen cells by a sensitive flow cytometric crossmatch method to examine their possible correlation with pregnancy outcome. Group 1 consisted of sera obtained from 24 women with a history of abortion, and lymphocytes and semen samples collected from their husbands at the same time of visiting the in vitro fertilization (IVF) Clinic at King Faisal Specialist Hospital and Research Center. Sera, lymphocytes and semen samples were also collected from six couples with no history of abortion who served as controls (Group 2). RESULTS: Using a sensitive flow cytometric assay to analyse the samples, without knowledge of clinical status, elevated levels of both IgG and IgM were detected in Group 1. However, no significant association was found when compared with normal females who had healthy pregnancies. CONCLUSION: Flow cytometry is a highly sensitive and specific tool for the detection of alloantibodies in human sera from patients with rejected transplanted organs. Our findings suggest that maternal antipaternal antibodies with respect to IgG and IgM classes do not play a major role in women with a history of recurrent abortions, despite the presence of increased levels of antibodies against three different sources of paternal antigens.