R.T. Scott

Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos

Abstract STUDY QUESTION What is the predictive value of trophectoderm mitochondrial DNA (mtDNA) quantity for blastocyst reproductive potential? SUMMARY ANSWER This study demonstrates that, within a given cohort, mtDNA quantitation does not distinguish between embryos that implant and embryos that do not implant after double embryo transfer (DET). WHAT IS ALREADY KNOWN An association between implantation failure and increased quantities of mtDNA has been observed in two studies but not in a third. STUDY DESIGN, SIZE AND DURATION A total of 187 patients (nine who received donor oocytes) with DET of one male and one female euploid blastocyst were included in this retrospective study, with 69 singleton deliveries providing the primary dataset to evaluate the predictive value of mtDNA for reproductive potential between January 2010 and July 2016. PARTICIPANTS/MATERIALS, SETTING AND METHOD MtDNA was quantified in cell lines to validate the quantitative PCR assay on limited quantities of starting material and then applied to 374 blastocyst biopsies. Pregnancies resulting in a singleton outcome were analyzed and newborn gender was utilized as a means to identify the implanted embryo. MtDNA quantity was then compared between implanted and non-implanted embryos in order to define the predictive value of mtDNA content for reproductive potential in this subset of patients. MAIN RESULTS AND THE ROLE OF CHANCE An initial comparison of mtDNA levels between all successful and unsuccessful embryos revealed no significant differences. In order to control for patient-specific variables, gender was subsequently used to identify the implanted embryo in DETs resulting in a singleton (n = 69). No systematic difference in relative mtDNA quantity was detected between implanted and non-implanted embryos. LIMITATIONS, REASONS FOR CAUTION This study was conducted at a single center and did not evaluate the entire cohort of embryos from each patient to evaluate cohort specific variation in mtDNA quantity. Although the largest of its kind so far, the sample size of DETs leading to a singleton was relatively small. WIDER IMPLICATIONS OF THE FINDINGS These data highlight the importance of control over patient-specific variables when evaluating candidate biomarkers of reproductive potential. All current available data suggest that mtDNA quantification needs further study before its clinical use to augment embryo selection. STUDY FUNDING/COMPETING INTERESTS The authors have no potential conflict of interest to declare. No external funding was obtained for this study. TRIAL REGISTRATION NUMBER Not applicable.

Validation of a targeted next generation sequencing-based comprehensive chromosome screening platform for detection of triploidy in human blastocysts

Triploidy accounts for ~2% of natural pregnancies and 15% of cytogenetically abnormal miscarriages. This study aimed to validate triploidy detection in human blastocysts, its frequency and parental origin using genotyping data generated in parallel with chromosome copy number analysis by a targeted next generation sequencing (tNGS)-based comprehensive chromosome screening platform. Phase 1: diploid and triploid control samples were blinded, sequenced by tNGS and karyotype predictions compared for accuracy. Phase 2: tNGS was used to calculate the frequency of triploidy in 18,791 human blastocysts from trophectoderm (TE) biopsies. Phase 3: parental origin of the inherited extra alleles was evaluated by sequencing parental gDNA to validate triploidy predictions from Phase 2. All karyotypes and ploidy in controls from Phase 1 were correctly predicted by two independent methods. A blastocyst triploidy frequency of 0.474% (89/18,791) was observed in Phase 2 of the study. Finally, five suspected triploid blastocysts with parental DNA available were confirmed to be triploid and of maternal origin. tNGS provides higher sequencing depth in contrast to other contemporary NGS platforms, allowing for accurate single nucleotide polymorphism calling and accurate detection of triploidy in TE biopsies. Triploidy in intracytoplasmic sperm injection-derived blastocysts is rare and mostly of maternal origin.

SNP Array, qPCR, and Next-Generation Sequencing-Based Comprehensive Chromosome Screening

Comprehensive chromosome screening (CCS) has now demonstrated improvement to the success of IVF in multiple randomized controlled trials. Enhanced safety of embryo biopsy at the blastocyst stage and the ability to assess aneuploidy accurately for all 24 chromosomes are amongst the primary reasons for this recent success. Through the development of three unique methods of CCS, a number of important development and validation strategies have emerged. In addition, new information regarding some remaining challenges, including mosaicism and which patient populations may or may not benefit, has been obtained. This chapter discusses these new methodologies and strategies of validation and identifies important future directions for the appropriate use of CCS as a means to enable routine single euploid embryo transfer.

Hematologic Disease in Implantation Failure

Successful embryo implantation involves a number of events that occur at the microvascular level. Hematologic pathology, specifically inherited and acquired thrombophilias, has been proposed as a potential etiology for implantation failure. This idea has largely evolved from the robust body of literature surrounding thrombophilias and recurrent pregnancy loss. The impact of thrombophilias on recurrent implantation failure is less clear, although it is thought to have a similar underlying mechanism, namely, that microthrombosis causes disturbed blood flow to the endometrium and, in the case of recurrent implantation failure, impairs the initial vascularization process that is necessary for successful implantation to occur. In this chapter, the available literature regarding the potential role of thrombophilias in implantation failure and the evidence for possible therapeutic options will be reviewed.

Embryo and Endometrial Synchrony in Implantation Failure

Normal implantation requires synchronous timing between the endometrium and the embryo. A loss of this synchrony—termed dyssynchrony—occurs when the endometrium is not optimally receptive at the time the embryo is ready to implant. This issue related to timing may lead to implantation failure even when the endometrial is capable of being receptive and the embryo was capable of implantation and progressing through pregnancy to delivery. While the traditional view has represented dyssynchrony as pathology attributed singularly to the embryo or the endometrium, it is clear that both entities can have robust reproductive potential in isolation, and the issue lies in the combination of both of these entities at the appropriate time. The timing of the stimulus for secretory transformation may vary from cycle to cycle, and embryonic development can also vary with age. Thus, their respective contribution to dyssynchrony is not always reproducible from cycle to cycle—thus it may not be screened for in advance. All patients undergoing superovulation during IVF are at risk for embryonic-endometrial dyssynchrony based on timing when a critical level of progesterone is attained and the timing of embryonic blastulation.

Diminished ovarian reserve and poor response to stimulation in patients <38 years old: a quantitative but not qualitative reduction in performance

STUDY QUESTION Do infertile women aged <38 years with quantitative evidence of diminished ovarian reserve and/or poor response to stimulation also exhibit poor oocyte quality as measured by blastulation rates, aneuploidy rates, and live birth rates? SUMMARY ANSWER Young women with evidence of accelerated follicular depletion, either by precycle ovarian reserve testing or postcycle evidence of low oocyte yield, exhibit equivalent blastulation rates, aneuploidy rates and live birth rates per euploid embryo transfer as age-matched controls with normal precycle and postcycle parameters. WHAT IS KNOWN ALREADY Previous studies are conflicted as to whether women with evidence of diminished ovarian reserve and/or poor ovarian response are also at increased risk of exhibiting evidence of poor oocyte quality. Most prior studies have failed to adequately control for the confounding effect of female age on typical markers of oocyte quality in poor responders. The rate of follicular depletion occurs at around 38 years on average; thus, evidence of quantitative depletion before this would indicate a premature diminution of ovarian reserve and allow evaluation of whether markers of oocyte quality are tied to quantitative markers. STUDY DESIGN, SIZE, DURATION This was a retrospective cohort study at a single center between 2012 and 2016. This time frame was specifically chosen as all embryos were cultured to the blastocyst stage at this center during the study period (no cleavage stage transfers were performed). Two comparisons were made: precycle assessment of ovarian reserve (based on anti-mullerian hormone (AMH) level) and postcycle oocyte yield results. For each comparison, patients in <10th percentile were compared to patients in the interquartile range (IQR) with respect to blastulation rate, aneuploidy rate and live birth rate. A mixed effects model was created to control for female age (in the <38 year old range) and correlation among oocytes from a given cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS For the precycle blastulation analysis, only patients with AMH data available were included (345 patients with AMH in the <10th percentile versus 1758 patients with AMH in the 25th to 75th percentile (IQR)). To compare aneuploidy rates, the subset of these patients who pursued preimplantation genetic testing for aneuploidy (PGT-A) was then analyzed (124 patients in the <10th percentile versus 782 patients in the IQR). For the postcycle blastulation analysis, all patients who proceeded to retrieval (whether or not they also had AMH data available) were included (535 patients with oocyte yield in the <10th percentile versus 2675 patients in the IQR). To compare aneuploidy rates, the subset of these patients who pursued PGT-A was then analyzed (156 patients in the <10th percentile versus 1100 patients in the IQR). MAIN RESULTS AND THE ROLE OF CHANCE The adjusted odds of a given fertilized oocyte developing to a blastocyst, being aneuploid or leading to a live birth after euploid transfer were no different if the oocyte was retrieved from a cycle with ovarian reserve parameters or oocyte yield in the <10th percentile compared to an oocyte retrieved in a cycle with those parameters in the 25-75th percentile. An AMH level in the <10th percentile did more commonly result in cycle cancellation prior to retrieval and after retrieval prior to transfer due to global arrest of embryos. LIMITATIONS, REASONS FOR CAUTION The timing of retrieval in patients with fewer oocytes may be more optimal given the greater ability to discern the overall maturity of the cohort, thus enhancing performance per retrieved oocyte. Analyses included only first cycles. Subsequent adjustment of protocol due to prior performance may mean that some patients in the <10th percentile for oocyte yield are actually better prognosis patients than their first cycle indicates. Data on whether or not patients were on oral contraceptives at time that AMH level drawn was not available. Other unknown biases are also likely to be present given retrospective nature of the study. WIDER IMPLICATIONS OF THE FINDINGS While young women with evidence of quantitative depletion of ovarian reserve have lower live birth rates per stimulation cycle, this not attributable to poor oocyte quality because the blastulation rate per fertilized oocyte and live birth rate per embryo transfer are equivalent to that in women with normal quantitative markers of ovarian reserve. Thus, the pathophysiology mediating a premature quantitative decline in ovarian reserve appears different than that which mediates markers of oocyte quality, such as aneuploidy. Young poor responders may use this information to help guide embryo accumulation strategies when considering their family building plans. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

Validation of simultaneous diagnosis of single gene disorder (SGD) and next generation sequencing (NGS) - based comprehensive chromosomal aneuploidy screening (CCS) from a single trophectoderm (TE) biopsy

OBJECTIVE: We previously established a concurrent methodology of SGD pre-implantation genetic diagnosis (PGD) and CCS using quantitative real-time (q)PCR from the same TE biopsies, which resulted in excellent clinical outcomes. To improve the accuracy of CCS and reduce the cost, we switched the CCS platform from qPCR to NGS. This study sought to validate simultaneously testing SGD and CCS based on NGS from a single TE biopsy.

Preimplantation Genetic Diagnosis and Genetic Screening

There have been great advances in molecular screening and diagnostic techniques in the field of assisted reproductive technology and many of these advances have come in the field of reproductive genetics. Broadly, preimplantation genetic diagnostics can be utilized to identify a normal embryo amongst those which contain a single gene disorder (preimplantation genetic diagnosis—PGD) or can be utilized to identify embryos that have gained or are missing large segments of DNA or whole chromosomes (preimplantation genetic screening—PGS ). Both PGD and PGS have a wide variety of applications and can used to identify embryos which have the highest potential of forming a normal, healthy offspring. There are challenges in this process including safely obtaining genetic material in the form of an embryo biopsy as well as appropriately and accurate application of amplification and analysis strategies. With the advent of proper validation and utilization, both PGD and PGS have been shown to be useful in improving reproductive outcomes and will be discussed in this chapter.

DHEA supplementation can result in assay changes which may impact clinical decisions in IVF

Dear Editor, We read with interest Tsui et al.’s letter to the editor regarding DHEA-S interference with progesterone assays in reference to our manuscript. In it, they pointed out that the units for DHEA-S should have been micrograms per deciliter as noted in Fig. 2 of the manuscript. The micrograms per deciliter units should have been used throughout the manuscript and the use of micrograms per milliliter was an error, both in the scientific abstract and in the manuscript. DHEA-S controls from the manufacturer were supplied in micrograms per deciliter units and yielded assay results in the reportable range in micrograms per deciliter. With the proper units of measurement, the DHEA-S levels measured in our patient population, both those taking DHEA supplementation and controls, were within physiological ranges. We appreciate their careful review of the manuscript and appreciate the opportunity to correct this error. The authors repeated the experiment on their Siemens ADVIA Centaur assay and showed an increase in measured progesterone from 0.4 to 1.0 ng/mL which was a similar, but less pronounced, change noted in our manuscript in Fig. 1. There was a greater change noted on the progesterone assay of other platforms. The interactions reported with the corrected units of micrograms per deciliter rather than micrograms per milliliter remain true. Tsui et al.’s experiment lends support to the theory that circulating DHEA-S in women on supplementation may alter serum progesterone levels. Although the interactions may be small in absolute units, when clinical decisions regarding whether to cryopreserve all embryos rather than perform a fresh embryo transfer due to premature progesterone rise are in the balance, slight changes may have a meaningful clinical impact.

Characterization of reproductive endocrinology and infertility (REI) fellowship applicants: guiding our mentees toward success

BackgroundAdvanced subspecialty training in reproductive endocrinology and infertility (REI) entails a competitive application process with many data points considered. It is not known what components weigh more heavily for applicants. Thus, we sought to study the REI fellow applicant and compare 1) those who apply but do not receive an interview, 2) those who receive an interview but do not match, and 3) those who successfully match.MethodsThis retrospective cohort study was conducted at a single REI fellowship program from 2013 to 2017. Academic variables assessed included standardized test scores and total number of publications listed on their curriculum vitae. Logistic regression models were constructed to determine variables that were predictive of being offered an interview in our program and of matching in any program.ResultsThere were 270 applicants, of which 102 were offered interviews. Interviewed applicants had significantly higher mean USMLE 1 and CREOG scores, as well as total publications and total abstracts. There was no difference in Step 2 and Step 3 scores or in number of book chapters. Of those interviewed, USMLE scores remained predictive of matching in any program; however, publications and scientific abstracts were no longer predictive.ConclusionsThe decision to offer applicants interviews appears to be influenced by objective standardized test scores, as well as a threshold of academic productivity. These items are less predictive of matching once the interview process begins, indicating that other factors, such as performance during the interview day, may be more heavily weighted.