Euploid blastocysts implant irrespective of their morphology after NGS-(PGT-A) testing in advanced maternal age patients.

PURPOSE: Does blastocyst morphology following euploid elective single embryo transfer (eSET) after preimplantation genetic testing for aneuploidies (PGT-A) via next generation sequencing impact clinical outcome? METHODS: Two hundred ninety-six patients underwent PGT-A. Of 1549 blastocysts, 1410 blastocysts had a conclusive result after PGT-A and were included for analysis. An eSET policy was followed in a frozen embryo replacement cycle. A total of 179 euploid blastocysts were thawed and transferred. Clinical outcomes were categorized in four different embryo quality groups: excellent, good, average and poor. RESULTS: Euploidy rate was 19/36 (52.7%, 95% CI 37-68), 199/470 (42.3%, 95% CI 38-47), 156/676 (23.0%, 95% CI 20-26) and 39/228 (17.1%, 95% CI 13-23) in the excellent, good, average and poor quality blastocyst groups, respectively. Fitted logistic regression analysis taking into account the following covariables: female, age, embryo chromosomal status and day of blastocyst development/biopsy showed that morphology was predictive of the comprehensive chromosome screening result (p < 0.05). A logistic regression analysis was also performed on clinical outcomes taking into account the effect of blastocyst morphology and day of blastocyst development/biopsy. None of the parameters were shown to be significant, suggesting morphology and day of blastocyst development/biopsy do not reduce the competence of euploid embryos (p > 0.05). CONCLUSIONS: After eSET, implantation rate was 80-86%; live birth rate per embryo transfer was 60-73% and clinical miscarriage rate was found to be < 10% and were not significantly affected by the embryo morphology. Results are concordant with those reported when using aCGH and highlights the competence of poor-quality euploid embryos.

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

PURPOSE: The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage. METHODS: This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test. RESULTS: This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p < 0.05). Correlation analyses showed that there was both inverse and direct association between miRNAs and their target mRNAs. CONCLUSIONS: miRNAs target many mRNAs including ones involved in DNA repair mechanisms. This study suggests that miRNAs and their target mRNAs involved in DNA repair are expressed in preimplantation embryos. Similar to the miRNAs expressed in adult tissues, these miRNAs seem to have regulatory roles on their target DNA repair mRNAs during preimplantation embryo development.

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.

Factors influencing the outcome of intrauterine insemination (IUI): age, clinical variables and significant thresholds.

The aim was to investigate the influence of various biological factors upon the outcome of intrauterine insemination (IUI). The total IUI history (856 cycles) of 352 couples was studied. Live-birth showed a strong negative correlation with female age but no correlation with male age. Antimüllerian hormone (AMH) and antral follicle count (AFC) correlated negatively with female age, and follicle stimulating hormone (FSH) correlated positively. Significant thresholds were found for all three variables, and also for total motile count (TMC) in the prepared sperm. Calculating pregnancy losses per positive pregnancy showed a strong correlation with increasing female age. This was highly significant for biochemical losses but not for fetal heart miscarriages. Male age had no effect on rate of pregnancy loss. In conclusion, female age, FSH, AMH and TMC are good predictive factors for live-birth and therefore relate to essential in vivo steps in the reproductive process.

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors.

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.

Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt lucrative. Analysis of gametes prior to the initiation of an IVF cycle may improve the quality of embryos transferred. The clinical and scientific community considers it a matter of urgency to translate the basic science behind how a cell prepares for fertilization into routine clinical practice. However, it is equally important, if not more, to allow the science behind such applications to draw level with its practice before its widespread implementation.

The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia.

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia.

Assisted conception in women of advanced maternal age.

A delay in childbearing to later in life has increased the number of women of advanced maternal age (AMA) opting for assisted reproduction. Women should be made aware that there are age-related changes to fertility, including a decline in oocyte reserve and quality, in addition to an increase in the number of oocyte chromosomal aberrations. Success rates of assisted reproductive technology (ART) cycles decrease with advanced maternal age. There are different fertility options for women of AMA, including fertility preservation (oocyte or embryo freezing), in vitro fertilisation (IVF treatment) with or without preimplantation genetic screening and oocyte or embryo donation. Detailed counselling needs to be offered to these women with regard to the risks, success rates, ethical and legal implications of these fertility treatment options. Women of AMA should be screened for underlying medical conditions that could have an impact on maternal and neonatal morbidity and mortality.

Fall in implantation rates following ICSI with sperm with high DNA fragmentation.

BACKGROUND: There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy. METHODS: The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm. RESULTS: The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients. CONCLUSIONS: These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.

Clinical outcomes of a vitrified donor oocyte programme: A single UK centre experience.

OBJECTIVE: To assess the survival rate of vitrified oocytes used in an egg recipient programme and compare the clinical outcomes of pregnancy and live-birth rates per warmed oocyte with fresh autologous oocytes. The differences in the obstetrical outcomes between the two groups were also studied. DESIGN: A prospective case control study from a single in-vitro fertilisaton (IVF) Centre in UK SETTING: Centre of Reproductive and Genetic Health (CRGH), London POPULATION: Vitrified oocytes from egg donors and autologous fresh oocytes from patients attending for an IVF cycle METHODS: The study group consisted of 1490 vitrified oocytes, which were obtained from 145 egg donors who underwent a stimulation cycle at CRGH Centre. The control group included 145 age-matched women who underwent intra cytoplasmic sperm injection (ICSI) treatment with their own oocytes (n = 1528). The clinical outcomes clinical pregnancy rates (CPR) and live-birth rates (LBR) and obstetrical outcomes (gestational age and weight at delivery) were compared between the two groups. Statistical analysis of the summary data and logistic regression analysis was performed using statistical packages (SPSS Version 23 and Stata 2015). The percentages of all parameters in the cases and control groups were compared by Fisher's exact test. A statistical significance level of 5% was adopted throughout the study. MAIN OUTCOME MEASURES: Survival rate per thawed oocyte, clinical pregnancy rate and live-birth rate per embryo transfer was compared to the autologous oocyte group RESULTS: The survival rate of vitrified oocytes was 73.6% (95% CI: 71.3-75.8%). The clinical pregnancy rate (per embryo transfer) using vitrified oocytes was found to be 51.8% compared to 59.3% in the control group. The live birth rate per embryo transfer in the vitrified oocyte group was 46% (95% CI 37.4-54.7%) compared to 57.1% (95% CI 48.5-68.5%) in the control group. The live-birth rate per thawed oocyte was found to be 4.2%. The gestational ages of the fetus at delivery in both the groups were comparable 39.0 (95% CI 32.7-41.9%) and 39.1 (95% CI 25.6-42.0) (p = 0.38). There was no statistically significant difference in the birth weight between the study and the control group 3100 g (750-4337) and 3232 g (1616-4500) respectively (p = 0.28). CONCLUSIONS: This is the first study reporting on the efficacy of a vitrified donor oocyte programme from within the UK. There were no significant differences in the obstetrical outcomes between vitrified donor oocytes and autologous oocytes. The above data will be encouraging for women who are undertaking egg freezing for medical and or social reasons.

Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application.

From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources.

Ovum donation--a simplified approach.

To date, ovum donation (OD) has involved luteinizing hormone (LH) synchronization between recipient and donor for normally cycling women, and a complex steroid replacement regimen given on a sequential and incremental basis for women with primary or secondary ovarian failure. The authors designed a simple hormonal regimen applicable to both normally cycling women starting early in the cycle, and to those with ovarian failure. It consists of administering 2 mg estradiol (E2) valerate orally three or four times daily, augmented with either 100 mg progesterone (P) in ethyl oleate intramuscularly daily or 100 mg oral progesterone (P) orally three times daily, starting on the day preceding the recovery of the donated oocytes. Gamete intrafallopian transfer procedure was undertaken for women with patent tubes and in vitro fertilization for those with obstructed tubes. The authors report their preliminary experience with 17 women who underwent ovum donation.

Basal 17beta-estradiol did not correlate with ovarian response and in vitro fertilization treatment outcome.

OBJECTIVE: To verify the correlation of basal 17beta-E(2) with ovarian response to stimulation and outcome of in vitro fertilization (IVF). DESIGN: Retrospective observational study. SETTING: The Assisted Conception Unit, University College London Hospitals. PATIENT(S): Three hundred five women undergoing IVF and IVF with intracytoplasmic sperm injection. INTERVENTION(S): Basal follicle-stimulating hormone (FSH) and 17beta-E(2) were assessed. The cutoff level for day 2 E(2) established was 250 pmol/L. Each patient was noted for below (group A) or above (group B) the cutoff point according to her basal E(2) level. MAIN OUTCOME MEASURE(S): Basal E(2), age, duration of infertility, cycle day 2 FSH, number of ampules of gonadotropin used, number of days of stimulation, number of retrieved oocytes, fertilization rate, number of embryos transferred, number of cycles with embryo freezing, cancellation rate, clinical pregnancy rate, and implantation rate were compared between the two groups. RESULT(S): No differences were found between group A and group B in the number of oocytes retrieved (8.8 +/- 4.2 vs. 9.3 +/- 4.8), embryos transferred (2.5 +/- 0.8 vs. 2.7 +/- 0.7), cancellation (9.1% vs. 6.9%), pregnancy (24.8% vs. 30%), and implantation rate (12.3% vs. 15.6%). Correlation coefficient and coefficient of determination showed no significant correlation between basal E(2) and the number of oocytes retrieved, age, and basal FSH. CONCLUSION(S): In our study population, basal E(2) was not a sensitive predictor of ovarian response to stimulation and did not correlate with IVF outcome.

Unexplained infertility--the value of Pergonal superovulation combined with intrauterine insemination.

Sixty-two women with unexplained infertility were studied. Fifteen (group 1) had timed intrauterine insemination (IUI), 25 (group 2) were treated by Pergonal (Serono Laboratories, Ltd., Welwyn Garden City, England) superovulation, and 22 (group 3) underwent Pergonal superovulation combined with IUI. Where Pergonal treatment was followed by insemination, a significantly greater pregnancy rate per cycle (P less than 0.05) was achieved, whether this group of patients was compared with those treated by IUI alone or with those treated with Pergonal alone. Moreover, the pregnancy rate in group 3 was comparable to that reported following gamete intrafallopian transfer (GIFT). The authors therefore suggest this form of treatment for patients with unexplained infertility prior to their referral to the more invasive procedure of GIFT.

Simultaneous evaluation of basal follicle-stimulating hormone and 17 beta-estradiol response to gonadotropin-releasing hormone analogue stimulation: an improved predictor of ovarian reserve.

OBJECTIVE: To compare different predictors of ovarian reserve. DESIGN: Prospective study. SETTING: The Assisted Conception Unit, University College London Hospitals. PATIENT(S): One hundred seventy-seven patients undergoing IVF treatment. INTERVENTION(S): Blood samples were collected on cycle day 2 to determine basal levels of FSH and 17beta-E2 and the FSH/LH ratio, and on cycle days 3 and 4 to assess the increase in FSH (deltaFSH) and 17beta-E2 (deltaE2) after the commencement of GnRH analogue (GnRH-a) stimulation. Ultrasound scans were performed during ovarian stimulation to assess the number of follicles. MAIN OUTCOME MEASURE(S): Day 2 FSH and 17beta-E2 levels, the FSH/LH ratio, and the deltaFSH and deltaE2 after the commencement of GnRH-a stimulation were correlated with the number of follicles obtained after ovarian stimulation. RESULT(S): All the possible predictors considered, except for the day 2 E2 level and the deltaFSH, correlated significantly with the ovarian response. The best single correlation was between the number of follicles and the deltaE2 (GnRH-a test). When the FSH level was evaluated simultaneously, the correlation was strengthened, resulting in a better negative predictive value. CONCLUSION(S): Simultaneous evaluation of basal levels of FSH and of the response of E2 to GnRH-a stimulation seems to be the best marker of ovarian reserve and a sensitive predictor of response to ovarian stimulation in patients undergoing IVF treatment.

[Heterotopic ovarian pregnancy after in-vitro fertilization and embryo transfer and contralateral tubal pregnancy after gamete intrafallopian transfer]. / Gravidanza eterotopica ovarica dopo fertilizzazione in vitro ed embryo transfer e gravidanza tubarica controlaterale dopo trasferimento intratubarico dei gameti.

One case of ovarian ectopic pregnancy associated with twin intrauterine viable pregnancy after IVF-ET, and one case of contralateral tubal ectopic pregnancy after GIFT performed with normal fallopian tubes, are reported. The possible pathogenesis and management are discussed.