Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes.

RESEARCH QUESTION: Can cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DESIGN: Cumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. RESULTS: A total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell-cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. CONCLUSIONS: These findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

A multicenter, prospective, blinded, nonselection study evaluating the predictive value of an aneuploid diagnosis using a targeted next-generation sequencing-based preimplantation genetic testing for aneuploidy assay and impact of biopsy.

OBJECTIVE: To determine the predictive value of an aneuploid diagnosis with a targeted next-generation sequencing-based preimplantation genetic testing for aneuploidy (PGT-A) assay in prognosticating the failure of a successful delivery. DESIGN: Prospective, blinded, multicenter, nonselection study. All usable blastocysts were biopsied, and the single best morphologic blastocyst was transferred before genetic analysis. Preimplantation genetic testing for aneuploidy was performed after clinical outcome was determined. Clinical outcomes were compared to PGT-A results to calculate the predictive value of a PGT-A aneuploid diagnosis. SETTING: Fertility centers. PATIENT(S): Couples undergoing their first in vitro fertilization cycle without recurrent pregnancy loss, antral follicle count < 8, or body mass index ≥ 35 kg/m2. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcome was the ability of the analytical result of aneuploid to predict failure to deliver (clinical result). A secondary outcome was the impact of the trophectoderm biopsy on sustained implantation. RESULT(S): Four hundred two patients underwent 484 single, frozen, blastocyst transfers. The PGT-A aneuploid diagnosis clinical error rate was 0%. There was no difference in sustained implantation between the study group and an age-matched control group, where biopsy was not performed (47.9% vs. 45.8). CONCLUSION(S): The PGT-A assay evaluated was highly prognostic of failure to deliver when an aneuploid result was obtained. Additionally, the trophectoderm biopsy had no detectable adverse impact on sustained implantation. CLINICAL TRIAL REGISTRATION NUMBERS: NCT02032264 and NCT03604107.

Evaluation of fertilization, usable blastocyst development and sustained implantation rates according to intracytoplasmic sperm injection operator experience.

RESEARCH QUESTION: Is intracytoplasmic sperm injection (ICSI) operator experience associated with fertilization, usable blastocyst development and sustained implantation rates (SIR) when at least two embryologists carry out ICSI for a single cohort of oocytes? DESIGN: A retrospective cohort study of all IVF/ICSI cycles at a single large infertility centre between 2008 and 2018. Cycles were included if a cohort of oocytes was split between two embryologists for ICSI. The embryologist's experience of ICSI was used to evaluate laboratory and clinical outcomes overall and by pairs of inseminating embryologists. Logistic regression, analysis of variance and Kruskal-Wallis testing were used where appropriate. RESULTS: Analysis of 14,362 ICSI procedures showed an association between least ICSI experience and lower mean fertilization rates (P < 0.0001), higher odds of failed fertilization (adjusted OR 4.3; P < 0.0001) and lower number of fertilization 'wins' per cohort (P < 0.0001). Usable blastocyst development rates (number of usable blastocysts/number of two pronuclear zygotes) were not associated with ICSI embryologist experience (P = 0.44), but the odds of obtaining no usable blastocysts were higher (adjusted OR 1.4; P < 0.0001) and the proportion of usable blastocyst 'wins' was lower (P = 0.0001) when embryologists with the least experience carried out ICSI. Increased ICSI experience was associated with higher mean SIR (P < 0.0001). Laboratory and clinical outcomes were similar among embryologists once 1000 ICSI cycles and above were carried out. CONCLUSIONS: Increased ICSI operator experience is associated with higher fertilization rates, SIR and a lower likelihood of failed fertilization and usable blastocyst development. Splitting a single oocyte cohort between more than one embryologist for ICSI is a quality-control measure that can be implemented.

Comparison of pregnancy outcomes following intrauterine insemination in young women with decreased versus normal ovarian reserve.

OBJECTIVE: To evaluate pregnancy outcomes following intrauterine insemination (IUI) in young women with low ovarian reserve compared to age-matched controls. DESIGN: Retrospective cohort SETTING: Single infertility center (July 2001-August 2018) PATIENT(S): Patients <35 years of age undergoing at least one IUI cycle with a documented serum anti-Müllerian hormone (AMH) level, patent fallopian tubes, and total motile sperm count of ≥10 million at the time of IUI. INTERVENTION(S): None MAIN OUTCOME MEASURE(S): The primary outcome was the presence of a positive serum hCG pregnancy test (>2 mIU/mL) obtained 2 weeks after the IUI procedure. Secondary outcomes included the incidence of live birth, biochemical loss, clinical miscarriage, and ectopic pregnancy. Additionally, cumulative reproductive outcomes including up to seven IUI cycles were calculated and compared between groups. RESULTS: A post-hoc power calculation demonstrated that the study sample size yielded >80% power to detect a 7% difference between groups in the primary outcome. There were 3019 patients included: 370 with AMH <1.0 ng/mL and 2649 with AMH ≥1.0 ng/mL. When adjusting for IUI treatment strategy, number of dominant follicles at time of IUI and body mass index, no difference in per-cycle or cumulative reproductive outcomes was identified between patients with low AMH (<1.0 ng/mL) and normal AMH (≥1.0 ng/mL). Analyses by treatment strategy also showed no difference in reproductive outcomes. CONCLUSION: Young patients (<35 years of age) with diminished ovarian reserve conceived as often and had per-cycle and cumulative pregnancy outcomes similar to those of age-matched controls after IUI, regardless of treatment strategy.

Embryo's Natural Motion (enMotion): a paired randomized controlled trial evaluating a dynamic embryo culture system.

OBJECTIVE: To determine if a dynamic embryo culture system affects the reproductive potential of human embryos resulting from in vitro fertilization (IVF). DESIGN: Paired randomized controlled trial (RCT). SETTING: IVF center. PATIENT(S): IVF patients with normal ovarian reserve eligible for two-embryo transfer. INTERVENTION: IVF care was routine until fertilization was confirmed. Two-pronuclear embryos (2PNs) were then randomized: One-half of each patient's 2PNs were cultured in dynamic culture and one-half in static culture. Preimplantation genetic testing for embryonic aneuploidy was used to control for aneuploidy and allow for DNA fingerprinting. The best euploid blastocyst from each culture system was selected and patients underwent a frozen two-embryo transfer. If a singleton gestation resulted, DNA-fingerprinting was used to determine which of the two blastocysts implanted. The dynamic platform used was the NSSB-300 (Nepagene). MAIN OUTCOME MEASURE(S): The primary outcome was the proportion of usable blastocysts obtained. The secondary outcome was sustained implantation rate (SIR). RESULT(S): One hundred participants completed oocyte retrieval and blastocyst vitrification for frozen-thawed embryo transfer; 609 dynamic 2PNs and 615 static 2PNs were followed; and 304 blastocysts developed in dynamic culture and 333 blastocysts developed in static culture. In the paired analysis, the rate of usable blastulation was similar between dynamic and static culture (58.3% vs. 57.1%). In addition, there was no difference in the rate of aneuploidy (20.0% vs. 33.3%) or SIR (67.1% vs. 63.1%) between groups. CONCLUSION(S): In this paired RCT, dynamic culture did not improve usable blastulation rate or SIR. CLINICAL TRIAL REGISTRATION NUMBER: NCT02467725.

Sperm Mitochondrial DNA Copy Number Is Not a Predictor of Intracytoplasmic Sperm Injection (ICSI) Cycle Outcomes.

This study is to determine if sperm mitochondrial DNA copy number (mtDNA CN) is associated with fertilization, blastulation, blastocyst euploidy, and live birth rates in in vitro fertilization (IVF) with ICSI cycles. This is a cohort study conducted on stored sperm samples which were collected prospectively and used to create blastocysts transferred in a couple's first ICSI transfer cycle between 2007 and 2013 at a single large infertility center. Samples from ICSI cycles utilizing surgical or cryopreserved sperm or day 3 embryo biopsy were excluded. The primary outcome was live birth rate. Secondary outcomes included fertilization, usable blastocyst development, and blastocyst euploidy rates. Unique sperm samples used to create transferred embryos were identified. Mitochondrial DNA CN was evaluated using TaqMan® quantitative real-time polymerase chain reaction (qPCR) assays normalized to a nuclear control for relative quantitation. Linear regression and mixed effects logistic regression used were appropriate. A total of 2062 unique sperm samples used to create transferred embryos were included. Lower relative sperm mtDNA content was associated with increased pre-wash sperm motility (p < 0.001). No significant association was identified between sperm mtDNA CN and fertilization (p = 0.40), usable blastocyst development (p = 0.36), blastocyst euploid (p = 0.10), and live birth rates (p = 0.42) while adjusting for sperm pre-wash motility and maternal age. Sperm mtDNA CN is not prognostic of fertilization, usable blastocyst development, euploidy and live birth rates in an infertile population undergoing IVF with ICSI.

Clinical error rates of next generation sequencing and array comparative genomic hybridization with single thawed euploid embryo transfer.

We investigated clinical error rates with single thawed euploid embryo transfer (STEET) diagnosed by next generation sequencing (NGS) and array comparative genomic hybridization (aCGH). A total of 1997 STEET cycles after IVF with preimplantation genetic testing for aneuploidy (PGT-A) from 2010 to 2017 were identified; 1151 STEET cycles utilized NGS, and 846 STEET cycles utilized aCGH. Any abortions, spontaneous or elective, in which products of conception (POCs) were collected were reviewed. Discrepancies between chorionic villus sampling, amniocentesis, or live birth results and PGT-A diagnosis were also included. Primary outcomes were clinical error rate per: ET, pregnancy with gestational sac, live birth, and spontaneous abortion with POCs available for analysis. Secondary outcomes included implantation rate (IR), spontaneous abortion rate (SABR), and ongoing pregnancy/live birth rate (OPR/LBR). The clinical error rates in the NGS cohort were: 0.7% per embryo, 1% per pregnancy with gestational sac, and 0.1% rate per OP/LB. The error rate per SAB with POCs was 13.3%. The IR was 69.1%, the OPR/LBR was 61.6%, and the spontaneous abortion rate was 10.2%. The clinical error rates in the aCGH cohort were: 1.3% per embryo, 2% per pregnancy with gestational sac, and 0.4% rate per OP/LB. The error rate per SAB with POCs was 23.3%. The IR was 63.8%, the OPR/LBR was 54.6%, and the SAB rate was 12.4%. Our findings demonstrate that, although NGS and aCGH are sensitive platforms for PGT-A, errors still occur. Appropriate patient counseling and routine prenatal screening are recommended for all patients undergoing IVF/PGT-A.

Impact of paternal age on embryology and pregnancy outcomes in the setting of a euploid single-embryo transfer with ejaculated sperm: retrospective cohort study.

OBJECTIVE: To evaluate the impact of paternal age on embryology and pregnancy outcomes in the setting of a euploid single-embryo transfer. DESIGN: Retrospective cohort study. SETTING: Not applicable. PATIENTS: Couples undergoing a first in vitro fertilization cycle with fresh ejaculated sperm who used intracytoplasmic sperm injection for fertilization followed by preimplantation genetic testing for aneuploidy and single-embryo transfer of a euploid embryo between January 2012 and December 2018. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Embryology outcomes assessed were fertilization rate, blastulation rate, and euploid rate. Pregnancy outcomes assessed included positive human chorionic gonadotropin rate, delivery rate, biochemical loss rate, and clinical loss rate. RESULTS: A total of 4,058 patients were assessed. After adjusting for female age, increased paternal age in the setting of fresh ejaculated sperm use was associated with decreased blastulation and decreased euploid rate using 40 years as an age cutoff. CONCLUSIONS: In this study, advancing paternal age appears to have a detrimental impact on rates of blastocyst formation and euploid status. However, if a euploid embryo is achieved, older paternal age does not appear to affect negatively pregnancy outcomes.

Consistency in rates of diagnosis of embryonic mosaicism, segmental abnormalities, and "no call" results among experienced embryologists performing preimplantation genetic testing for aneuploidy.

OBJECTIVE: To determine whether differences exist in rates of subchromosomal abnormalities, mosaicism, and "no call" results among embryologists performing and loading trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A). DESIGN: Retrospective cohort. SETTING: Large infertility center. PATIENTS: All patients undergoing in vitro fertilization with PGT-A. INTERVENTIONS: The NexCCS next generation sequencing platform was used for PGT-A. The χ2 testing assessed differences in rates of primary outcomes between embryologists. Intraclass correlation coefficients evaluated inter-embryologist reliability in rates of abnormal and no call results. Median absolute performance difference (MAPD) scores, which quantify the impact of technical variation on analytical performance, were averaged for individual embryologists. Analysis of variance assessed differences in mean MAPD scores. MAIN OUTCOME MEASURES: Interoperator variability in rates of mosaic, segmental, and no call results. RESULTS: Four embryologists performed 30,899 biopsies and 6 embryologists loaded specimens into designated tubes. Among individuals performing trophectoderm sampling, rates of mosaicism were 4.3% to 6.1%, segmental errors were 9.0% to 10.7%, and inconclusive results were 1.1% to 2.9%. For those loading, the incidence of mosaicism was 4.2% to 5.9%, subchromosomal abnormalities was 9.7% to 10.4%, and no call results was 1.2% to 2.2%. The intraclass correlation coefficient was 0.978 for embryologists performing biopsies and 0.981 for those loading. Differences in mean MAPD scores were within 0.6% and 0.2% of each other for doing biopsies and loading embryologists, respectively. CONCLUSIONS: Rates of mosaicism, segmental, and no call PGT-A results are consistent among experienced embryologists. Due to the large sample size included, differences within 1% of the mean were deemed clinically irrelevant despite statistical significance.

PROGNOSTIC VALUE OF BLASTOCYST GRADE AFTER FROZEN EUPLOID EMBRYO TRANSFER IN PATIENTS WITH RECURRENT PREGNANCY LOSS.

OBJECTIVE: To determine if trophectoderm (TE) grade or inner cell mass (ICM) grade have predictive value after euploid frozen embryo transfer (euFET) among RPL patients. DESIGN: Retrospective cohort study. SETTING: Single fertility center, 2012-2018. PATIENTS: Patients with ≥ 2 prior pregnancy losses performing PGT-A with ≥1 euploid embryo for transfer. INTERVENTIONS: All patients underwent ICSI, trophectoderm biopsy, blastocyst grading and vitrification, and single euFET. Outcome of the first transfer was recorded. MAIN OUTCOME MEASURES: Live birth (LB) and clinical miscarriage (CM) rates. RESULTS: 660 euFET were included. In a binomial logistic regression analysis accounting for age, BMI, AMH and day of blastocyst biopsy, ICM grade C was not significantly associated with odds of live birth (aOR 0.50, 95% CI 0.24-1.02 p=0.057), miscarriage (aOR 1.67, 95% CI 0.56-5.00, p=0.36) or biochemical pregnancy loss (aOR 1.58, 95% CI 0.53-4.75, p=0.42). TE grade C was significantly associated with odds of live birth (aOR 0.49, 95% CI 0.28-0.86, p=0.01) and was not associated with odds of miscarriage (aOR 2.00, 95% CI 0.89-4.47, p=0.09) or biochemical pregnancy loss (aOR 1.85, 95% CI 0.77-4.44, p=0.17). Blastocyst grade CC had significantly lower LB rate compared to all other blastocyst grades (p<0.05, chi-square analysis). CONCLUSION: Embryo grade CC and TE grade C are associated with decrease in odds of LB after euFET in RPL patients. Embryo grade is not associated with odds of CM in this cohort of RPL patients, suggesting that additional embryonic or uterine factors may influence risk of pregnancy loss.

Total Motile Sperm Count Trend Over Time: Evaluation of Semen Analyses From 119,972 Men From Subfertile Couples.

OBJECTIVE: To determine whether a clinically-relevant change in the total motile sperm count (TMSC) over time exists within the subfertile population. METHODS: The first semen analysis of all men presenting to selected infertility centers in 2 countries between 2002 and 2017 were evaluated. Semen analyses were categorized into 3 clinically-relevant groups based on treatment options: TMSC >15 million (M) (Group 1), in which no insemination intervention would be required; TMSC 5-15 M (Group 2), in which intrauterine insemination would be appropriate; and TMSC of <5 M (Group 3), in which in vitro fertilization would be considered. Relationships between male age, TMSC, trend of TMSC, and TMSC group membership by year were assessed. RESULTS: A total of 119,972 first semen analyses were included. The proportion of men with normal TMSC (>15 M) was found to decline approximately 10 percentage points over the past 16 years in the analysis of combined centers (odds ratio 0.967; 95% confidence interval = 0.963-0.971; P = 2.2e-16). A reciprocal increase was distributed between both the moderate (5-15 M) and severe (<5 M) oligozoospermia groups. Additionally, TMSC declined 1.1 percentage points with each year of advancing paternal age. No difference was seen in age at presentation by year. CONCLUSION: The proportion of men with normozoospermia declined and that of men at risk of requiring fertility treatment increased over the study time period. Although several unknown factors may have influenced our data as a result of the retrospective design, a shift in treatment group membership over time may be clinically relevant.

Interactive case-based learning improves resident knowledge and confidence in reproductive endocrinology and infertility.

Resident physicians' scores on the REI section of the CREOG exam are traditionally low, and nearly 40% of house staff nation-wide perceive their REI knowledge to be poor. We aimed to assess whether an interactive case-based group-learning curriculum would narrow the REI knowledge gap by improving understanding and retention of core REI concepts under the time constraints affecting residents. A three-hour case-based workshop was developed to address four primary CREOG objectives. A multiple-choice test was administered immediately before and after the intervention and 7 weeks post-workshop, to evaluate both knowledge and confidence. Following the intervention, residents self-reported increased confidence with counseling and treatment of PCOS, ovulation induction cycle monitoring, counseling and treatment of POI, and breaking bad news related to infertility (p < 0.05). The multiple-choice exam was re-administered 7 weeks post-intervention, and scores remained significantly improved compared to pre-workshop scores (p < 0.05). At that time, all residents either strongly agreed (91.7%) or agreed (8.3%) that the case-based interactive format was preferable to traditional lecture-based teaching. In conclusion, a nontraditional curriculum aimed at teaching core REI concepts to residents through interactive case-based learning can be successfully integrated into a residency curriculum, and significantly improves knowledge and confidence of critical concepts in REI.

Paternal Age Is Not Associated With Pregnancy Outcomes After Single Thawed Euploid Blastocyst Transfer.

Although controversial, increasing paternal age has been shown to negatively affect assisted reproductive technology (ART) outcomes and success rates. Most studies investigating the effect of paternal age on ART outcomes use a donor oocyte model to minimize maternal aneuploidy contribution. This study sought to determine whether increasing paternal age is associated with adverse in vitro fertilization (IVF) outcomes when aneuploidy is minimized using preimplantation genetic screening. There were 573 single thawed euploid embryo transfers from 473 patients undergoing oocyte donor and autologous IVF cycles. Cycles were categorized according to paternal age at oocyte retrieval, and an age adjustment was performed for maternal age in order to evaluate for an isolated paternal age effect. Fertilization rate was found to decrease significantly with increasing paternal age ( P = .04). After controlling for oocyte age, there was no significant difference in pregnancy outcomes across all paternal age categories after euploid embryo transfer, including implantation rate ( P = .23), clinical pregnancy rate ( P = .51), and spontaneous abortion rate ( P = .55). Therefore, if a couple is able to produce and transfer a single thawed euploid embryo, no difference in IVF pregnancy outcomes is identified with increasing paternal age.

FMR1 gene CGG repeat variation within the normal range is not predictive of ovarian response in IVF cycles.

The relationship between FMR1 CGG premutation status and decreased ovarian responsiveness is well established. The association between FMR1 CGG repeat number in the currently defined normal range (less than 45 repeats) and ovarian reserve, however, is controversial. This retrospective study examined whether variation in CGG repeat number in the normal range was associated with markers of ovarian response in IVF cycles. The first IVF cycle of 3006 patients with FMR1 CGG repeat analysis was examined. Only patients carrying two alleles with less than 45 CGG repeats were included for analysis. The CGG repeat number furthest from the modal peak was plotted against number of mature oocytes retrieved and no correlation was identified. Patients were also separated into biallelic genotype groups, based on the recently proposed narrower "new normal" range of 26-34 CGG repeats. A linear regression showed that none of the biallelic genotype groups were associated with a decreased oocyte yield. The euploidy rates after comprehensive chromosomal screening were equivalent among the genotype groups. No difference was found in the rate of cycle cancellation for poor response. Despite increasing use, FMR1 CGG repeats in the normal range cannot be used as a predictor of ovarian response to gonadotrophin stimulation.

Discrepant diagnosis rate of array comparative genomic hybridization in thawed euploid blastocysts.

PURPOSE: Preimplantation genetic screening (PGS) and diagnosis (PGD) with euploid embryo transfer is associated with improved implantation and live birth rates as compared to routine in vitro fertilization. However, misdiagnosis of the embryo is a potential risk. The purpose of this study was to investigate the clinical discrepant diagnosis rate associated with transfer of trophectoderm-biopsied blastocysts deemed to be euploid via array comparative genomic hybridization (aCGH). METHODS: This is a retrospective cohort study including cycles utilizing PGS or PGD with trophectoderm biopsy, aCGH, and euploid embryo transfer at a large university-based fertility center with known birth outcomes from November 2010 through July 2014 (n = 520). RESULTS: There were 520 embryo transfers of 579 euploid embryos as designated by aCGH. Five discrepant diagnoses were identified. Error rate per embryo transfer cycle was 1.0 %, 0.9 % per embryo transferred, and 1.5 % per pregnancy with a sac. The live birth (LB) error rate was 0.7 % (both sex chromosome errors), and the spontaneous abortion (SAB) error rate was 17.6 % (3/17 products of conception tested, but could range from 3/42 to 7/42). No single gene disorders were mistakenly selected for in any known cases.  CONCLUSIONS: Although aCGH has been shown to be a highly sensitive method of comprehensive chromosome screening, several possible sources of error still exist. While the overall error rate is low, these findings have implications for counseling couples that are contemplating PGS and PGD with aCGH.