Exposure to phthalates in couples undergoing in vitro fertilization treatment and its association with oxidative stress and DNA damage.

This prospective study of 599 couples seeking fertility treatment and who were recruited between 2015 and 2017 was conducted to (a) explore the associations between phthalate exposure and in vitro fertilization (IVF) outcomes; and (b) examine the implication of oxidative stress as a mediator of these. We measured eight phthalate metabolites in two spot urine samples; oxidative stress biomarkers such as malondialdehyde, 8-hydroxy-2-deoxyguanosine, hydrogen peroxide, catalase (CAT), and total antioxidant capacity in follicular fluid and seminal plasma. We also examined DNA damage in sperm and granulosa cells. Couples were exposed to a broad range of phthalate compounds and seven metabolites were detected in over 94% of the urine samples, whereas monobenzyl phthalate was found in only 24% of women and 26% of men. Our results showed high levels of seven urinary phthalate metabolites (except monobenzyl phthalate) and a notable increase in many oxidative stress markers in both follicular fluid and seminal plasma. However, their associations with exposure were rather limited. Multivariate binomial regression modeling showed higher levels of follicular CAT levels reduced the probability of fertilization rate (≤ 50%) [Adjusted relative risk (RRadj) = 0.52, p = 0.005] and unsuccessful live birth (RRadj = 0.592, p = 0.023). We observed a 46% decrease in the probability of clinical pregnancy in association with an elevated percentage of DNA in the tail (RRadj = 0.536, p = 0.04). There was a 32% and 22% increase in the probability of clinical pregnancy and unsuccessful live birth associated with higher levels of mono-(2-ethylhexyl) phthalate (RRadj = 1.32, p = 0.049) and monoethyl phthalate (RRadj = 1.22, p = 0.032) in women, respectively. In contrast, the probability of clinical pregnancy reduced by 20% with higher levels of mono-(2-ethyl-5-carboxypentyl) phthalate (RRadj = 0.797, p = 0.037) and 19.6% with mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) (RRadj = 0.804, p = 0.041) in men. Other oxidative stress biomarkers or urinary phthalate metabolites showed suggestive relationships with certain IVF outcomes. Lastly, our results demonstrated that elevated levels of CAT in follicular fluid might have a positive impact on fertilization rate ≥ 50% and successful live birth. CAT seems to play a potential role in mediating the relationship between the risk of poor fertilization rate and MEOHP and mono-isobutyl phthalate. Additional data are required to understand the clinical implications of oxidative stress and its contribution to the reproductive toxicity of phthalate exposure.

The extent and predictors of phthalate exposure among couples undergoing in vitro fertilization treatment.

Phthalates are chemicals used as plasticizers and solvents in many consumer products but are suspected of disrupting the endocrine system and are known for their reproductive/developmental health risks. This study examined the extent and predictors of phthalate exposure among 599 couples undergoing in vitro fertilization. A questionnaire was administered to obtain sociodemographic, health, and lifestyle data, and two spot urine samples were collected from the couples to analyze eight phthalate metabolites, cotinine (COT) as a smoking index, and creatinine to adjust for urine dilution. Seven phthalate metabolites were detected in > 94% of the urine samples, and monobenzyl phthalate (MBzP) was found in 24% of the women and 26% of their male partners. Median phthalate levels were highest for monoethyl phthalate (MEP), at 333.26 µg/l in women and 290 µg/l in male partners, and lowest for MBzP, at 1.17 µg/l in women and 1.14 µg/l in male partners. Correlation coefficients of ≥ 0.4 between the women and their male partners for the eight urinary phthalate metabolites may indicate a shared source of exposure. A multivariate regression model was used to assess the association between predictors and each urinary phthalate metabolite. Several potential predictors for the variations in specific urinary phthalate metabolites were identified, including the body mass index, age, socioeconomic status, and regional distribution for both women and their male partners but with slightly different patterns. Women with a history of breastfeeding, using bottled water for cooking and storing food in plastic bags had lower MEP (8.7%), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) (9.2%), and both mono-iso-butyl phthalate and MECPP (8.2 and 8.1%). A history of contraceptive use was associated with an increase in MECPP (8.7%), mono-(2-ethyl-5-hydroxyhexyl) phthalate (11.4%), mono-(2-ethyl-5-oxohexyl) phthalate (7.6%), and the molar sum of bis (2-ethylhexyl) phthalate metabolites (8.9%). Urinary COT levels were associated with an increase of 10-16% in all urinary metabolites in women but of only 10.5% in mono-(2-ethylhexyl) phthalate in male partners. More than 95% of the couples reported the use of cosmetics, perfumes, and personal-care products, but we were not able to find associations with urinary phthalate metabolites, perhaps due to their short half-lives. MEP levels associated with the use of household cleaning products were 11.2% higher in male partners. Our levels were generally higher than those reported elsewhere, perhaps due to different lifestyles, cultural practices, dietary habits, use of personal-care products, and governmental legislation.

Gene expression profiling of granulosa cells from PCOS patients following varying doses of human chorionic gonadotropin.

PURPOSE: Human chorionic gonadotrophin (hCG) has been used to induce ovulation and oocyte maturation. Although the most common dose of hCG used in IVF is 10,000 IU, there are reports that suggest 5,000 IU is sufficient to yield similar results. The objective of this study is to evaluate the dose dependent differences in gene expression of granulosa cells following various doses of hCG treatment. METHODS: Patients with polycystic ovarian syndrome (PCOS) were stimulated for IVF treatment. The hCG injection was either withheld or given at 5,000 or 10,000 IU. Granulosa cells from the follicular fluids have been collected for RNA isolation and analyzed using Affymetrix genechip arrays. RESULTS: Unsupervised hierarchical clustering based on whole gene expression revealed two distinct groups of patients in this experiment. All untreated patients were clustered together whereas hCG-treated patients separated to a different group regardless of the dose. A large number of the transcripts were similarly up- or down-regulated across both hCG doses (2229 and 1945 transcripts, respectively). However, we observed dose-dependent statistically significant differences in gene expression in only 15 transcripts. CONCLUSIONS: Although hCG injection caused a major change in the gene expression profile of granulosa cells, 10,000 IU hCG resulted in minimal changes in the gene expression profiles of granulosa cells as compared with 5,000 IU. Thus, based on our results, we suggest the use of 10,000 IU hCG should be reconsidered in PCOS patients.

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.

Oxidative stress and DNA damage status in couples undergoing in vitro fertilization treatment.

This study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde, 8-hydroxy-2-deoxyguanosine, hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in the sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. Separate multivariate linear regression was used to assess associations between levels of each oxidative stress biomarker and each hormone and smoking parameter (modeled as natural log-transformed). The findings indicate that some oxidative stress and DNA damage biomarkers played a role in disrupting certain reproductive hormones in women and their male partners either by overproducing reactive oxygen species or reducing antioxidant defense capacity. Although women were nonsmokers, COT levels > 50 and 10 µg/L in urine and follicular were observed in 5.7 and 1.7%, respectively. Levels of follicular fluid COT were positively associated with H2O2 and TM. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate > 50%, biochemical pregnancy, clinical pregnancy, and live birth). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that the elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%). The higher antioxidant activity (CAT and TAC) in follicular fluid might positively impact specific IVF outcomes. LAY SUMMARY: Oxidative stress occurs when antioxidant molecules are insufficient in the body to destroy free radicals that can damage the cells, proteins and DNA, causing different health conditions, including infertility. The role of oxidative stress in female infertility has not received as much attention as male infertility, and research is still limited. This study explored whether the overproduction of free radicals can impact the success of in vitro fertilization (IVF) treatment using several biological markers such as hydrogen peroxide, catalase, and total antioxidant capacity. Our findings revealed that the high antioxidant levels in the fluid surrounding the egg were linked with a high fertilization rate. Additionally, oxidative stress status in couples was associated negatively with several reproductive hormones and smoking status. Biomarkers of oxidative stress and DNA damage might have potential applications in evaluating IVF patients' clinical characteristics such as causes of infertility, hormonal profile, fertilization rate, implantation and live birth.

Infertility treatment outcome in sub groups of obese population.

BACKGROUND: Obesity is a common disorder with a negative impact on IVF treatment outcome. It is not clear whether morbidly obese women (BMI >= 35 kg/m2) respond to treatment differently as compared to obese women (BMI = 30-34.9 kg/m2) in IVF. Our aim was to compare the outcome of IVF or ICSI treatments in obese patients to that in morbidly obese patients. METHODS: This retrospective cohort study was conducted in a tertiary care centre. Patients inclusion criteria were as follows; BMI >or= 30, age 20-40 years old, first cycle IVF/ICSI treatment with primary infertility and long follicular pituitary down regulation protocol. RESULTS: A total of 406 obese patients (group A) and 141 morbidly obese patients (group B) satisfied the inclusion criteria. Average BMI was 32.1 +/- 1.38 kg/m2 for group A versus 37.7 +/- 2.99 kg/m2 for group B. Patient age, cause of infertility, duration of stimulation, fertilization rate, and number of transferred embryos were similar in both groups. Compared to group A, group B had fewer medium size and mature follicles (14 vs. 16), fewer oocytes collected (7 vs. 9) and required higher doses of HMG (46.2 vs. 38.5 amps). There was also a higher cancellation rate in group B (28.3% vs. 19%) and lower clinical pregnancy rate per started cycle (19.9% vs. 28.6%). CONCLUSION: In a homogenous infertile and obese patient population stratified according to their BMI, morbid obesity is associated with unfavorable IVF/ICSI cycle outcome as evidenced by lower pregnancy rates. It is recommended that morbidly obese patients undergo appropriate counseling before the initiation of this expensive and invasive therapy.

Couples exposure to phthalates and its influence on in vitro fertilization outcomes.

This prospective study examined the associations between the levels of eight urinary phthalate metabolites in 599 couples and in vitro fertilization (IVF) outcomes. We used log-binomial multivariate regression to estimate relative risks (RR) for the association between phthalate concentration and IVF binary outcomes (fertilization rate >50%, biochemical pregnancy, clinical pregnancy and live birth) for each woman after adjusting the model for the concentration in a male partner and each relevant confounders. RR was expressed per unit increase in log-transformed urinary metabolite concentration. The percentage of bis-2-ethylhexyl phthalate (DEHP) metabolites excreted as mono-2-ethylhexyl phthalate (MEHP) was calculated as %MEHP. Urinary MEHP in women was associated with an increased risk of biochemical pregnancy (RR = 1.35; p = 0.04), failed clinical pregnancy (RR = 1.56; p = 0.006) and live birth (RR = 1.54; p = 0.011). An increase in monoethyl phthalate was associated with a high risk of failed clinical pregnancy (RR = 1.25; p = 0.03) and live birth (RR = 1.35; p = 0.006). An increase in %MEHP was associated with an increase in the risk of biochemical pregnancy (RR = 1.55; p = 0.05), failed clinical pregnancy (RR = 1.73; p = 0.02) and live birth (RR = 1.65; p = 0.046). Our results demonstrated that exposure to some phthalates may adversely affect IVF outcomes, particularly when couples' exposure was jointly modeled, emphasizing the importance of a couple-based approach in assessing fertility outcomes. The associations between IVF outcomes and DEHP metabolites were stronger in women whose %MEHP was >75th percentile which may be due to their less efficient metabolism and excretion of DEHP and/or MEHP.

The relationships between urinary phthalate metabolites, reproductive hormones and semen parameters in men attending in vitro fertilization clinic.

Evidence from previous studies has shown that phthalates may play a role in male reproductive function; however, results are still inconclusive, and the mechanism remains unclear. In this study, we first assessed whether exposure to phthalates is associated with altered reproductive hormones and semen parameters in 599 men attending an in vitro fertilization clinic. Secondly, we evaluated whether reproductive hormones could play a mediating role in the association between phthalates and sperm parameters. Eight phthalate metabolites were measured in two different spot urine samples: mono­n­butyl phthalate, mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP), monobenzyl phthalate, and four oxidative metabolites of di­(2­ethylhexyl) phthalate (DEHP) [i.e., mono­(2­ethylhexyl) phthalate (MEHP), mono­(2­ethyl­5­hydroxyhexyl) phthalate (MEHHP), mono­(2­ethyl­5­oxohexyl) phthalate (MEOHP), and mono­(2­ethyl­5­carboxypentyl) phthalate (MECPP)]. Semen parameters (concentration, volume, motility, and morphology) and reproductive hormones, i.e., follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone, estradiol (E2), testosterone (TEST) and prolactin (PROL) were also determined and considered the main study outcomes. Separate multivariate linear regression was used to assess associations between levels of each urinary phthalate metabolite, molar sum of DEHP metabolites (∑DEHP), percentage of MEHP to ∑DEHP (%MEHP), and each outcome (natural log-transformed). Inverse associations were observed between TEST and MiBP (ß = -0.099), FSH and MEHHP (ß = -0.087), and PROL and MEOHP (ß = -0.102), while a positive relationship was seen between E2 and MEP (ß = 0.098). %MEHP was associated positively with FSH (ß = 0.118) and LH (ß = 0.099), but negatively with TEST/LH (ß = -0.086) and TEST/E2 (ß = -0.109). Sperm concentration was associated positively with MECPP (ß = 0.131), MEHHP (ß = 0.117), MEOHP (ß = 0.107) and ∑DEHP (ß = 0.111), but negatively with %MEHP (ß = -0.135). All p-values were <0.05. Sobel's test indicated that FSH mediated significantly up to 60% of the positive relationship between sperm concentration and MEHHP, while FSH and LH mediated respectively 15 and 12% of the inverse association between sperm concentration and %MEHP. Further research on this topic is warranted.

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

BACKGROUND: To 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. METHODS: A retrospective cohort study including all patients who had IVF-ET from January 2003-December 2005 conducted at a tertiary center. RESULTS: A 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). CONCLUSION: There 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.

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.

TLE6 mutation causes the earliest known human embryonic lethality.

BACKGROUND: Embryonic lethality is a recognized phenotypic expression of individual gene mutations in model organisms. However, identifying embryonic lethal genes in humans is challenging, especially when the phenotype is manifested at the preimplantation stage. RESULTS: In an ongoing effort to exploit the highly consanguineous nature of the Saudi population to catalog recessively acting embryonic lethal genes in humans, we have identified two families with a female-limited infertility phenotype. Using autozygosity mapping and whole exome sequencing, we map this phenotype to a single mutation in TLE6, a maternal effect gene that encodes a member of the subcortical maternal complex in mammalian oocytes. Consistent with the published phenotype of mouse Tle6 mutants, embryos from female patients who are homozygous for the TLE6 mutation fail to undergo early cleavage, with resulting sterility. The human mutation abrogates TLE6 phosphorylation, a step that is reported to be critical for the PKA-mediated progression of oocyte meiosis II. Furthermore, the TLE6 mutation impairs its binding to components of the subcortical maternal complex. CONCLUSION: In this first report of a human defect in a member of the subcortical maternal subcritical maternal complex, we show that the TLE6 mutation is gender-specific and leads to the earliest known human embryonic lethality phenotype.

Poor prognosis in cycles following "genuine" empty follicle syndrome.

OBJECTIVES: The aim of this study was to investigate the prognosis in future IVF cycles of patients with empty follicle syndrome (EFS). STUDY DESIGN: EFS cases and their future cycles were reviewed. Clinical pregnancy rate per started cycle was taken as the primary outcome in assessing the future outcome in IVF treatment cycles. RESULTS: A total of 3023 patients underwent 5238 IVF treatment cycles. Twenty-six patients (1%) had a total of 58 (1%) cycles of EFS. Thirteen women went through 32 further IVF treatment cycles following the diagnosis of EFS, yielding only two clinical pregnancies, giving a clinical pregnancy rate of 6.25% per started cycle. In addition, four patients had recurrence in a total of 15 cycles. CONCLUSIONS: The occurrence of EFS will indicate poor IVF success in subsequent IVF cycles. Patients with "genuine EFS" should be counselled about the outcome of their future IVF cycles.

Preimplantation genetic diagnosis for Zellweger syndrome.

OBJECTIVE: To report on the first live birth of a normal child after performance of preimplantation genetic diagnosis (PGD) for Zellweger syndrome (ZS). DESIGN: Case report. SETTING: Tertiary-care hospital. PATIENT(S): A family with four children diagnosed with ZS, who were all born at term and who expired around 4 months of age. INTERVENTION(S): In vitro fertilization and preimplantation genetic diagnosis. MAIN OUTCOME MEASURE(S): Preimplantation genetic diagnosis of ZS in embryos, and live birth from the transferred normal embryos. RESULT(S): After PGD, two genotypically normal embryos were transferred back to the mother. Pregnancy ensued, and a healthy baby girl was delivered in week 40 of pregnancy. The baby was confirmed as genotypically wild-type, and free of any sign of ZS. CONCLUSION(S): To the best of our knowledge, this is the first successful PGD for ZS caused by mutation in PEX26 gene, with the subsequent delivery of a homozygous normal baby.

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.

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 rearrangements. 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.

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.

Y chromosome microdeletions: are they implicated in teratozoospermia?

BACKGROUND: Y chromosome microdeletions are known to impair spermatogenesis. Screenings for these microdeletions are performed mostly in patients with sperm count abnormalities. METHODS: We have screened the Y chromosome of 80 infertile patients with sperm morphological abnormalities. DNA from sperm, peripheral blood or single sperm following multiple displacement amplification (MDA) was utilized to amplify 20 specific sequence-tagged sites (STS) by PCR. RESULTS: Y chromosome microdeletions were detected in sperm DNA from four of the teratozoospermic patients; while none of the 53 men with normal sperm morphology had any deletions. Two of the four patients with deletions also provided peripheral blood and a fresh semen sample. Both patients had none of the STS deleted in the peripheral blood DNA. Y chromosome microdeletion analysis in the MDA amplified SRY-positive single sperm DNA confirmed the presence of the same deletion in all 10 sperm for one patient and eight out of 10 sperm in the second patient. CONCLUSIONS: Our observations suggest that some of the teratozoospermia might be related to gonadal mosaic Y chromosome microdeletions. Gonadal mosaicism can be a source of de novo transmissions of Y chromosome microdeletions. The application of MDA can yield enough DNA from a single sperm for genetic analyses.

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.

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.