Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility. RESULTS: Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing. CONCLUSIONS: Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Kinase insert domain receptor/vascular endothelial growth factor receptor 2 (KDR) genetic variation is associated with ovarian hyperstimulation syndrome.

BACKGROUND: The objective of this investigation was to determine if kinase insert domain/vascular endothelial growth factor receptor 2 (KDR/VEGFR2) genetic variation was associated with the development of ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation (COH). METHODS: This was a case-control study of 174 patients who underwent controlled ovarian stimulation. Patient blood samples were genotyped for single nucleotide polymorphisms (SNPs) spanning the KDR locus. OHSS development, clinical outcome variables, SNP and haplotype frequencies were compared between control (n = 155) and OHSS (n = 19) groups. RESULTS: Patients who developed OHSS had significantly higher response markers (estradiol levels of the day of hCG administration, number of follicles developed, number of eggs retrieved) than control patients. When adjusted for age and self-identified race, the rs2305945 G/T genotype was associated (P = 0.027) with a decreased risk (OR = 0.30; 95% CI = 0.10, 0.93) of developing OHSS using an overdominant model. The rs2305945 G/T variant was also associated with decreased COH response (number of follicles, number of eggs retrieved) in an overdominant model. The rs2305948, rs1870378, rs2305945 (C-T-G) haplotype was associated with both decreased COH response and OHSS risk (unadjusted OR = 0.10; 95% CI = 0.01, 0.80, P = 0.031). CONCLUSIONS: The KDR receptor is believed to play a central role OHSS development and is a target for pharmacological prevention of OHSS. These results indicate that genetic variation in the KDR gene may impact individual risk of developing OHSS from COH. In addition, the rs2305948 SNP and C-T-G haplotype might serve as potential biomarkers for poor ovarian response to COH.

Association between the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) rs4073366 polymorphism and ovarian hyperstimulation syndrome during controlled ovarian hyperstimulation.

BACKGROUND: The aim of this study was to determine the relationship between a purported luteinizing hormone/chorionic gonadotropin (LHCGR) high function polymorphism (rs4539842/insLQ) and outcome to controlled ovarian hyperstimulation (COH). METHODS: This was a prospective study of 172 patients undergoing COH at the Fertility and IVF Center at GWU. DNA was isolated from blood samples and a region encompassing the insLQ polymorphism was sequenced. We also investigated a polymorphism (rs4073366 G > C) that was 142 bp from insLQ. The association of the insLQ and rs4073366 alleles and outcome to COH (number of mature follicles, estradiol level on day of human chorionic gonadotropin (hCG) administration, the number of eggs retrieved and ovarian hyperstimulation syndrome (OHSS)) was determined. RESULTS: Increasing age and higher day 3 (basal) FSH levels were significantly associated with poorer response to COH. We found that both insLQ and rs4073366 were in linkage disequilibrium (LD) and no patients were homozygous for both recessive alleles (insLQ/insLQ; C/C). The insLQ variant was not significantly associated with any of the main outcomes to COH. Carrier status for the rs4073366 C variant was associated (P = 0.033) with an increased risk (OR 2.95, 95% CI = 1.09-7.96) of developing OHSS. CONCLUSIONS: While age and day 3 FSH levels were predictive of outcome, we found no association between insLQ and patient response to COH. Interestingly, rs4073366 C variant carrier status was associated with OHSS risk. To the best of our knowledge, this is the first report suggesting that LHCGR genetic variation might function in patient risk for OHSS.

Multinucleation of a sibling blastomere on day 2 suggests unsuitability for embryo transfer in IVF-preimplantation genetic screening cycles.

OBJECTIVE: To evaluate the impact of multinucleation of a sibling blastomere of day 2 embryos on the rate of aneuploidy detected by day 3 preimplantation genetic screening (PGS) biopsy and the effect on subsequent implantation and pregnancy rates. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): A total of 141 couples undergoing their first IVF-PGS cycle for idiopathic recurrent pregnancy loss (RPL) or multiple failed IVF implantations. INTERVENTION(S): Biopsy of single-nucleated blastomeres for PGS analysis of chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22 by fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Aneuploidy, implantation, and pregnancy rates. RESULT(S): PGS revealed an increased incidence of aneuploidy when comparing multinucleated day 2 embryos with single-nucleated embryos (85% vs. 78%; relative risk 0.92 (95% confidence interval 0.84-1.00). Transfer of single-nucleated euploid embryos resulted in clinical pregnancy and implantation rates of 28% and 24%. Transfer of multinucleated euploid embryos resulted in no implantations. CONCLUSION(S): The presence of multinucleated blastomeres on day 2 of embryo development, 1 day before biopsy, predicts an increase of aneuploidy and poor pregnancy outcomes in IVF-PGS cycles.

Day 2 embryo transfer (ET) and day 3 ET afford similar reproductive outcomes in the poor responder.

A retrospective review of 237 initial, fresh nondonor IVF cycles in which all embryos generated during the cycle were transferred on either day 2 (n = 109) or day 3 (n = 128) were evaluated with regards to reproductive outcomes. Patients who underwent a day 2 ET had similar conception (18% vs. 16%; relative risk [RR], 1.1; 95% confidence interval [CI], 0.64-1.95), clinical pregnancy (13% vs. 16%; RR, 0.8; 95% CI, 0.44-1.55), implantation (6% vs. 7%; RR, 0.9; 95% CI, 0.50-1.68), and live-birth (10% vs. 16%; RR, 0.7; 95% CI, 0.32-1.29) rates as those who underwent a day 3 ET.

Profound teratospermia does not influence sex chromosomal aneuploidy rate in in vitro fertilization-preimplantation genetic diagnosis cycles.

Because spermatocyte meiotic error results in embryonic sex chromosomal aneuploidy, it is speculated that teratospermia correlates with increased embryonic sex chromosomal abnormalities. Our findings contradict this theory, suggesting that morphology does not correlate with sex chromosomal genotype.

A novel surgical treatment for cesarean scar pregnancy: laparoscopically assisted operative hysteroscopy.

OBJECTIVE: To describe laparoscopically assisted hysteroscopy as a unique surgical intervention for a cesarean section scar ectopic pregnancy. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 44-year-old woman, pregnant at 5 weeks and 6 days' gestational age with a cesarean section scar ectopic pregnancy. INTERVENTION(S): The patient underwent serial transvaginal ultrasound examinations with Doppler flow studies, followed by a laparoscopically assisted operative hysteroscopy for evacuating the cesarean scar ectopic pregnancy. MAIN OUTCOME MEASURE(S): Conservation of the uterus, fertility preservation. RESULT(S): Successful conservative surgical treatment of cesarean section scar ectopic pregnancy. CONCLUSION(S): Conservative laparoscopically assisted operative hysteroscopy can be used successfully in hemodynamically stable patients with a cesarean section scar ectopic pregnancy.

The use of lead follicle diameter to initiate gonadotropin-releasing hormone antagonist does not affect in vitro fertilization clinical pregnancy, implantation, or live birth rates: a prospective, randomized study.

In this prospective, randomized study, waiting for the lead follicle to reach 14 mm before initiating GnRH antagonist administration effectively prevents an LH surge and ovulation during an IVF cycle. Furthermore, delaying GnRH start until the dominant follicle reaches 14 mm neither impacts the clinical pregnancy, implantation, or live birth rates nor increases the incidence of severe ovarian hyperstimulation syndrome.

Effects of triploidy after intracytoplasmic sperm injection on in vitro fertilization cycle outcome.

OBJECTIVE: To evaluate the impact on the rates of clinical pregnancy and live birth of polyploidy after intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): One hundred forty-three patients undergoing their first IVF-embryo transfer cycle requiring ICSI. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Patients were divided into two groups on the basis of the proportion of post-ICSI triploid fertilization that was observed at the time of fertilization assessment: group 1 included patients with 20% 3PN zygotes. The primary outcomes compared between groups were pregnancy and implantation rates; secondary outcome measures included clinical-pregnancy rate and live-birth rate per embryo transfer. RESULT(S): Pregnancy, implantation, clinical-pregnancy, and live-birth rates were significantly higher in the cohort of patients who had 20% of zygotes appearing triploid (relative risk [RR] for pregnancy, 2.4 [95% confidence interval {CI}, 1.22-4.77]; RR for implantation, 2.6 [95% CI, 1.17-5.56]; RR for clinical pregnancy, 2.8 [95% CI, 1.16-6.85]; and RR for live birth, 2.6 [95% CI, 1.06-6.38]). The proportion of 3PN zygotes after ICSI had a statistically significant inverse relationship to clinical-pregnancy rate. CONCLUSION(S): The proportion of triploid zygotes after ICSI serves as a negative prognostic indicator for IVF cycle outcome. This finding suggests that triploidy proportion is a surrogate marker of oocyte competence that represents the integrity of the oocytes in the entire recruited cohort. Such findings therefore may influence recommendations for embryo transfer number and freezing of supernumerary embryos.

Does ethnicity influence in vitro fertilization (IVF) birth outcomes?

OBJECTIVE: To determine if ethnicity influences IVF birth outcome. DESIGN: Retrospective cohort study. SETTING: University-based IVF program. PATIENT(S): All African American women (n = 71) and Caucasian women (n = 180) who underwent initial fresh, nondonor IVF/embryo transfer (ET) cycles between January 1, 2004 and December 31, 2005. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gonadotropin dose, duration of stimulation, peak estradiol levels, oocyte yield, implantation, clinical pregnancy, and live birth rates. RESULT(S): African American women generated significantly fewer embryos than Caucasian women (5.3 +/- 3.7 vs. 6.6 +/- 4.8) despite having similar ages, day 3 FSH, peak estradiol levels, length of stimulation, and number of oocytes retrieved. In addition, compared with Caucasian women, African American had significantly greater body mass indices (26.5 +/- 5.2 vs. 23.7 +/- 4.8) and required significantly more total gonadotropin (IU) (4,791 +/- 2,161 vs. 3,725 +/- 2,005) for ovarian stimulation. African American women were more likely to have uterine fibroids (21% vs. 3%) and tubal factor infertility (23% vs. 9%). Caucasian women were more likely to have unexplained infertility (53% vs. 32%). Differences in embryo yield between patient groups persisted after accounting for differences in infertility diagnosis and prevalence of fibroids. Biochemical, clinical pregnancy, and live birth rates as well as implantation rates (number of sacs visualized/number of embryos transferred) did not significantly differ between groups. CONCLUSION(S): Although African Americans yield fewer embryos than Caucasian women with IVF, these ethnic groups do not seem to differ with regard to IVF pregnancy outcomes.

The influence of sperm morphology on preimplantation genetic diagnosis cycles outcome.

OBJECTIVE: To evaluate the impact of abnormal sperm morphology on the rates of aneuploidy, implantation, and clinical pregnancy. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): Fifty-two patients undergoing their first IVF-preimplantation genetic diagnosis (PGD) cycle. INTERVENTION(S): The PGD analysis of embryos. MAIN OUTCOME MEASURE(S): Patients were divided into two groups based on sperm morphology: teratospermic group (TSG) and normal sperm group (NSG). The primary outcome measures of rates of aneuploidy, implantation, clinical pregnancy rate (PR) per cycle, and clinical PR per embryo transfer were compared between TSG and NSG according to PGD analysis results. RESULTS: A higher percentage of normal embryos was seen in the NSG (32%) versus the TSG (20%). Overall, 30% of IVF-PGD cycles had no normal embryos for transfer. The clinical PR per cycle was 44% in the NSG compared to 14% in the TSG (relative risk [RR] = 3.19; 95% confidence interval [CI] 1.1-9.0). A similar trend was noted with the clinical PR per embryo transfer with 57% patients becoming pregnant in the NSG versus 20% patients in the TSG (RR = 2.76; 95% CI 1.2-7.2). Implantation was twice as likely to occur in the NSG as compared to TSG (RR = 2.5; 95% CI 1.1-7.2). CONCLUSION(S): Rates of euploidy, implantation, clinical PR per cycle, and clinical PR per embryo transfer were higher in the NSG compared to the TSG, suggesting that sperm morphology plays an important role in the outcome of IVF-PGD cycles.

Novel follicular-phase gonadotropin-releasing hormone antagonist stimulation protocol for in vitro fertilization in the poor responder.

Poor responders continue to be vexing in infertility therapy. By using GnRH antagonists before ovarian stimulation, we demonstrate an improvement in oocyte, embryo, and zygote yield in patients with a prior poor response.

Second cycle: to hatch or not to hatch?

The objective of this study was to assess the impact of assisted hatching (AH) on pregnancy rate (PR), clinical pregnancy rate (CPR), and implantation rate (IR) after a single failed, fresh, nondonor IVF cycle. Accordingly, we report that patients with one prior implantation failure benefit from AH with improved PR, CPR, and IR in a subsequent cycle.