Do sperm factors influence embryonic aneuploidy? Long live the oocyte.

STUDY QUESTION: What is the impact of male age- and sperm-related factors on embryonic aneuploidy? SUMMARY ANSWER: Using a 3-fold analysis framework encompassing patient-level, embryo-level, and matching analysis, we found no clinically significant interactions between male age and sperm quality with embryo ploidy. WHAT IS KNOWN ALREADY: While the effect of maternal age on embryo chromosomal aneuploidy is well-established, the impact of male age and sperm quality on ploidy is less well-defined. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study analyzed autologous preimplantation genetic testing for aneuploidy (PGT-A) and frozen embryo transfer cycles from December 2014 to June 2021. The study involved 11 087 cycles from 8484 patients, with a total of 35 797 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: The aneuploidy rate, calculated as the ratio of aneuploid blastocysts to the total number of blastocysts biopsied in a single treatment cycle, was evaluated. In the embryo-level analysis, the main outcome measure was the ploidy state of the embryos. The study employed a multifaceted analytical approach that included a patient-level analysis using generalized linear mixed models, an embryo-level analysis focusing on chromosomal ploidy, and a propensity score matching analysis contrasting groups with distinct ploidy rates (0% and 100%). There were no interventions as this was an observational study of PGT-A cycles. MAIN RESULTS AND THE ROLE OF CHANCE: No clinically relevant factors influencing ploidy rate related to male and sperm quality were revealed. In contrast, female age (coefficient = -0.053), BMI (coefficient = 0.003), prior ART cycle (coefficient = -0.066), and number of oocytes retrieved (coefficient = -0.018) were identified at the patient level. Embryo analysis identified age (coefficient = -0.1244) and ICSI usage (coefficient = -0.0129) as significant factors. Despite these, no significant interactions between male and female assessed factors on the ploidy rate emerged. Propensity score matching between maximal (100% vs 0%) euploid rates did not reveal significant differences of influence by male age and sperm quality. LIMITATIONS, REASONS FOR CAUTION: The focus on patients having blastocyst biopsy for PGT-A may not reflect the broader IVF population. Other semen quality parameters like DNA fragmentation were not included. Exclusion of embryo mosaicism from the analysis could affect aneuploidy rate interpretations. There may also be unmeasured influences like lifestyle or environmental factors. WIDER IMPLICATIONS OF THE FINDINGS: Male age and sperm quality parameters were consistent across both maximal and minimal ploidy rate comparisons. No significant clinical characteristics related to the factors assessed for the male-influenced blastocyst ploidy status, confirming the dominancy of the oocyte and female age. STUDY FUNDING/COMPETING INTEREST(S): The study was not funded. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

STUDY QUESTION: Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? SUMMARY ANSWER: The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. WHAT IS KNOWN ALREADY: The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. STUDY DESIGN, SIZE, DURATION: As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 µl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. LIMITATIONS, REASONS FOR CAUTION: The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03520933).

Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

STUDY QUESTION: Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? SUMMARY ANSWER: Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. WHAT IS KNOWN ALREADY: Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. STUDY DESIGN, SIZE, DURATION: In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. TRIAL REGISTRATION NUMBER: N/A.

Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy.

Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.

ART outcomes in lean compared to obese phenotypes of polycystic ovarian syndrome.

RESEARCH QUESTION: To investigate differences in reproductive outcomes among IVF patients with lean compared to obese polycystic ovarian syndrome (PCOS) phenotypes. DESIGN: A retrospective cohort study of patients with PCOS who underwent IVF in a single, academically affiliated infertility center in the USA between December 2014 and July 2020. The diagnosis of PCOS was assigned based on Rotterdam criteria. Patients were designated as lean (< 25) or overweight/obese (≥ 25) PCOS phenotype based on BMI (kg/m2) at cycle start. Baseline clinical and endocrinologic laboratory panel, cycle characteristics, and reproductive outcomes were analyzed. The cumulative live birth rate included up to 6 consecutives cycles. A Cox proportional hazards model and Kaplan-Meier curve for estimating live birth rates were used to compare the two phenotypes. RESULTS: A total of 1395 patients who underwent 2348 IVF cycles were included. The mean (SD) BMI was 22.7 (2.4) in the lean and 33.8 (6.0) in the obese group (p < 0.001). A number of endocrinological parameters were similar between lean and obese phenotypes: total testosterone 30.8 ng/dl (19.5) vs 34.1 (21.9), p > 0.02 and pre-cycle hemoglobin A1C 5.33% (0.38) vs 5.51% (0.51) p > 0.001, respectively. The CLBR was higher in those with a lean PCOS phenotype: 61.7% (373/604) vs 54.0% (764/1414) respectively. Miscarriage rates were significantly higher for O-PCOS patients (19.7% (214/1084) vs 14.5% (82/563) p < 0.001) and the rate of aneuploids was similar (43.5%, 43.8%, p = 0.8). A Kaplan-Meier curve estimating the proportion of patients with a live birth was higher in the lean group (log-rank test p = 0.013). After adjusting for potential confounders, the lean phenotype was associated with an increased hazard ratio for live birth: HR = 1.38 p < 0.001. CONCLUSIONS: Lean PCOS phenotype is associated with a significantly higher CLBR compared to their obese counterparts. Miscarriage rates were significantly higher among obese patients, despite comparable pre-cycle HBA1C and similar aneuploidy rates in patients who underwent PGT-A.

Compared with conventional insemination, intracytoplasmic sperm injection provides no benefit in cases of nonmale factor infertility as evidenced by comparable euploidy rate.

OBJECTIVE: To evaluate whether differences in euploidy rates exist between intracytoplasmic sperm injection (ICSI) and conventional insemination (CI) in nonmale factor infertility cases. DESIGN: Retrospective cohort study. SETTING: A single, academically affiliated infertility center in the United States. PATIENTS: A total of 3554 patients who underwent in vitro fertilization cycles from January 2014 to December 2021. All cycles that had preimplantation testing for aneuploidy (PGT-A) performed by trophectoderm biopsy and had a postpreparation sperm concentration >4 million total motile sperm per milliliter were included. MAIN OUTCOME MEASURES: The primary outcome was the embryo euploidy rate per embryo biopsied in the ICSI vs. CI group. Secondary outcomes included the fertilization rate and number of embryos biopsied. Generalized estimating equations with a Poisson distribution were used to estimate the euploid rate ratio (with total embryos biopsied as an offset), while accounting for multiple retrievals per patient. To adjust for confounding, a propensity score model was fit for ICSI using 14 baseline female and male characteristics. RESULTS: Oocytes retrieved and the number of embryos biopsied were similar in both groups, while the fertilization rate per oocyte retrieved was significantly lower with ICSI (0.64 vs. 0.66). The proportion of euploid embryos in the ICSI group was significantly lower when compared with CI (0.47 vs. 0.52), with a euploid rate ratio of 0.89. Interestingly, when accounting for the variation in PGT reference laboratories over the study time period, adjusting for the date of procedure did not change the relationship between ICSI and euploid rate (rate ratio = 0.89); however, after adjusting for the PGT reference laboratory, the relationship between ICSI and euploid rate was no longer significant (rate ratio = 0.97). CONCLUSIONS: In the setting of nonmale factor infertility, ICSI resulted in a lower fertilization rate and an 11% lower embryo euploid rate compared with CI. Although the data are not statistically significant when adjusted for the PGT reference laboratory, we still can conclude that ICSI does not provide any benefit. These data support the recommendation that CI should be the preferred methodology for fertilization in nonmale factor infertility cases.

Modified natural and optimized programmed frozen embryo transfers have equivalent live birth rates: an analysis of 6,682 cycles.

OBJECTIVE: To compare the live birth rates (LBRs) in modified natural and programmed single blastocyst frozen embryo transfer (FET) cycles. DESIGN: Retrospective cohort study. SETTING: University-affiliated fertility practice. PATIENT(S): Patients who underwent single blastocyst FETs between January 2014 and December 2019. A total of 15,034 FET cycles from 9,092 patients were reviewed; 1,186 modified natural and 5,496 programmed FET cycles from 4,532 patients met the inclusion criteria for analysis. INTERVENTION(S): No intervention. MAIN OUTCOME MEASURE(S): The primary outcome measure was the LBR. RESULT(S): There was no difference in live birth after programmed cycles using intramuscular (IM) progesterone or a combination of vaginal progesterone and IM progesterone compared with that after modified natural cycles (adjusted relative risks, 0.94 [95% confidence interval {CI}, 0.85-1.04] and 0.91 [95% CI, 0.82-1.02], respectively). The relative risk of live birth decreased in programmed cycles that used exclusively vaginal progesterone compared with that in modified natural cycles (adjusted relative risk, 0.77 [95% CI, 0.69-0.86]). CONCLUSION(S): The LBR decreased in programmed cycles that used only vaginal progesterone. However, no difference in the LBRs existed between modified natural and programmed cycles if programmed cycles used either IM progesterone or a combination of IM and vaginal progesterone protocols. This study demonstrates that modified natural FET cycles and optimized programmed FET cycles have equivalent LBRs.

Identification of miR-34-3p as a candidate follicular phase serum marker for endometriosis: a pilot study.

OBJECTIVE: To identify early follicular phase microribonucleic acids (miRNAs) that are altered in serum of women with endometriosis. DESIGN: Case-control study. SETTING: Large university-affiliated in vitro fertilization center. PATIENT(S): Women with (n = 21) and without (n = 24) endometriosis. INTERVENTION(S): Serum samples were obtained from laparoscopy-confirmed patients with endometriosis. MAIN OUTCOME MEASURE(S): The differential expression of serum miRNAs relative to controls was measured using the NanoString nCounter technology and validated by quantitative real-time polymerase chain reaction in an independent cohort of 27 patients with endometriosis and controls (n = 24). Microribonucleic acid target signaling pathways and genes were analyzed bioinformatically. A chemically modified stable miR-34-3p oligonucleotide was used to examine the effect on proliferation of VK2E6/E7 endometrial cells in vitro. RESULT(S): Eighteen miRNAs were significantly up-regulated, and 1 miRNA (hsa-miR-34c-3p) was significantly down-regulated in the follicular phase of patients with endometriosis. The analysis of target signaling pathways using TargetScan predicted regulation of the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, Hippo, adenosine monophosphate-activated protein kinase, transforming growth factor beta, and endometrial cancer pathways, which have been implicated in the pathogenesis of endometriosis, by these miRNAs. The analysis of sequence complementarity identified prostaglandin E2 receptor 4, interleukin 6 signal transducer, and polo-like kinase 4 genes as possible direct targets of hsa-miR-34-3p. DSDI-1, a chemically modified stable miR-34-3p oligonucleotide, reduced cell proliferation in VK2E6/E7 endometrial cells in vitro. CONCLUSION(S): The follicular phase miRNA levels are altered in serum of women with endometriosis and may be useful as reproducible detection biomarkers for early diagnosis of endometriosis. hsa-miR-34-3p is significantly down-regulated in endometriosis, targets endometriosis genes, and reduces endometrial cell proliferation in vitro. These results support hsa-miR-34-3p as a potential therapeutic target in endometriosis.

A diagnosis of diminished ovarian reserve does not impact embryo aneuploidy or live birth rates compared to patients with normal ovarian reserve.

OBJECTIVE: To estimate the aneuploidy rates in young women with diminished ovarian reserve (DOR) before treatment and poor ovarian response (POR) postretrieval. DESIGN: Retrospective cohort study. SETTING: A single academically-affiliated fertility clinic. PATIENT(S): Autologous frozen embryo transfer cycles from December 2014 to June 2020 were reviewed. Demographic and clinical factors that impact outcomes were used for propensity score matching (PSM) in a ratio of 2:1 and 4:1 for preimplantation genetic testing for aneuploidy pre-cycle DOR and POR after stimulation, respectively. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Aneuploid rates, defined as the number of aneuploid blastocysts divided by the number of biopsied blastocysts per cycle. No euploid embryos to transfer, defined as all cohorts of embryos being aneuploid. RESULT(S): A total of 383 women diagnosed with DOR were compared with matched controls. Aneuploid rates did not differ significantly between the two groups (42.2% vs. 41.7%; RR = 1.06; 95% CI, 0.95-1.06). No differences were identified in live birth rates per transfer between women with and without DOR after euploid single-embryo transfers (56.0% and 60.5%, respectively). An additional PSM analysis to assess aneuploidy rates for patients with POR (<5 oocytes) vs. those without it, resulted in similar rates of aneuploidy between the two comparison groups (41.1% vs. 44%, R = 1.02; 95% CI, 0.91-1.14). The prevalence of cycles with "no euploid embryos" in the POR cohort was higher (26% vs. 13%); however, rates of cases with a single embryo available for biopsy were lower in the DOR group, relative to controls (11% vs. 31%). CONCLUSION(S): Young women diagnosed with DOR or POR exhibited equivalent aneuploidy rates and live birth rates per euploid embryo transfer in a large matched population, based on age, body mass index, and IVF cycle initiation. The lower percentage of cycles with no euploid embryo available for transfer in DOR and POR patients is because of the decreased total number of oocytes/developing embryos and not because of increased aneuploidy rates in these groups.

Endometrial compaction does not predict live birth in single euploid frozen embryo transfers: a prospective study.

STUDY QUESTION: Is there a relationship between endometrial compaction and live birth in euploid frozen embryo transfer (FET) cycles? SUMMARY ANSWER: Live birth rates (LBRs) were similar in both patients that demonstrated endometrial compaction or no compaction in single euploid FETs. WHAT IS KNOWN ALREADY: There has been increasing interest in the correlation between endometrial compaction and clinical outcomes but there has been conflicting evidence from prior investigations. STUDY DESIGN, SIZE, DURATION: This was a prospective observational study from 1 September 2020 to 9 April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was performed at a single, academically affiliated fertility center in which patients who had an autologous single euploid FET using a programmed or modified natural cycle protocol were included. All embryos had trophectoderm biopsy for preimplantation genetic testing for aneuploidy followed by vitrification at the blastocyst stage. Two ultrasound measurements of endometrial thickness (EMT) were obtained. The first measurement (T1) was measured transvaginally within 1 day of initiation of progesterone or ovulation trigger injection, and a second EMT (T2) was measured transabdominally at the time of embryo transfer (ET). The primary outcome (LBR) was based on the presence and proportion of compaction (percentage difference in EMT between T1 and T2). MAIN RESULTS AND THE ROLE OF CHANCE: Of the 186 participants included, 54%, 45%, 35%, 28% and 21% of women exhibited >0%, ≥5%, ≥10%, ≥15% and ≥20% endometrial compaction, respectively. Endometrial compaction was not predictive of live birth at any of the defined cutoffs. A sub-analysis stratified by FET protocol type (n = 89 programmed; n = 97 modified natural) showed similar results. LIMITATIONS, REASONS FOR CAUTION: There was the potential for measurement error in the recorded EMTs. The T2 measurement was performed transabdominally, which may cause potential measurement error, as it is generally accepted that transvaginal measurements of EMT are more accurate, though, any bias is expected to be non-differential. The sub-analysis performed looking at FET protocol type was underpowered and should be interpreted with caution. Our study, however, represents a pragmatic approach, as it allowed patients to avoid having to come in for an extra transvaginal ultrasound the day before or on the day of ET. WIDER IMPLICATIONS OF THE FINDINGS: Assessing endometrial compaction may lead to unnecessary cycle cancellation. However, further studies are needed to determine if routine screening for endometrial compaction would improve clinical outcomes. STUDY FUNDING/COMPETING INTEREST(S): No authors report conflicts of interest or disclosures. There was no study funding. TRIAL REGISTRATION NUMBER: NCT04330066.

Fluorescence lifetime imaging microscopy (FLIM) detects differences in metabolic signatures between euploid and aneuploid human blastocysts.

STUDY QUESTION: Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts? SUMMARY ANSWER: FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts. WHAT IS KNOWN ALREADY: Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts. STUDY DESIGN, SIZE, DURATION: This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini-Hochberg's false discovery rate, at a q-value of 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04). LIMITATIONS, REASONS FOR CAUTION: This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. There are no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The use of propensity score matching to assess the benefit of the endometrial receptivity analysis in frozen embryo transfers.

OBJECTIVE: To study the impact of the endometrial receptivity analysis (ERA) on live birth rates in frozen embryo transfer (FET) cycles. DESIGN: Retrospective cohort study. SETTING: A single, large, university-affiliated infertility practice. PATIENT(S): Autologous FET cycles between January 1, 2014, and June 30, 2019, were reviewed. Multiple covariates that impact outcomes were used for propensity score matching; 133 ERA patients were matched to 353 non-ERA patients. Patients were assigned to the ERA group if they had an ERA during treatment and underwent at least one "personalized" FET based on the ERA recommendations. INTERVENTION(S): No interventions administered. MAIN OUTCOME MEASURE(S): Live birth rates per cycle in the FET cycle after ERA compared with that of matched non-ERA patients. RESULT(S): The live birth rates for the ERA group, 49.62%, and the matched non-ERA group, 54.96%, (odds ratio 0.8074; 95% confidence interval, 0.5424-1.2018) were not significantly different, nor was a difference seen in subanalyses based on prior number of FETs or receptivity status. CONCLUSION(S): The ERA identifies a patient's putative window of implantation with the goal of improving synchrony with the embryo, thereby achieving higher live birth rates. This study used propensity score matching to control for multiple covariates in a heterogenous group of patients to compare live birth rates. There was no difference in the live birth rate in patients who underwent the ERA compared with that of those who did not.

Elevated serum progesterone during in vitro fertilization treatment and the risk of ischemic placental disease.

BACKGROUND: Elevated progesterone on the day of human chorionic gonadotropin (hCG) administration is associated with decreased live birth rates in IVF cycles. The association with adverse pregnancy outcomes is unknown. OBJECTIVES: Assess the association between serum progesterone on the day of hCG administration and the risk of ischemic placental disease [IPD; preeclampsia, placental abruption, and/or small for gestational age (SGA)]. METHODS: We conducted a retrospective cohort study of autologous fresh IVF cycles resulting in delivery between 2005 and 2018. All IVF procedures were conducted at a large, university-affiliated infertility center. Patients were divided into tertiles based on their serum progesterone level on the day of hCG administration; the lowest tertile served as the reference group. We identified pregnancies complicated by preeclampsia and placental abruption using ICD-9/10 codes and medical record review. We defined SGA as < 10th percentile using U.S. growth curves. RESULTS: The cohort included 166 deliveries in the lowest tertile of progesterone (0.2-0.73 ng/ml), 166 deliveries in the middle (0.64-1.05 ng/ml) and 167 deliveries in the highest tertile (1.05-5.6 ng/ml). Compared with the lowest tertile, the risk of IPD was greater in the middle (RR 1.6; 95% CI 1.1-2.5) tertile after adjustment for age, parity, number of oocytes retrieved, and estradiol. The highest tertile was also not associated with an increased risk of IPD. CONCLUSION: In an IVF population, elevated serum progesterone in the range of 0.64-1.05 ng/mL on the day of hCG administration was associated with a small increased risk of IPD.

Multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes.

RESEARCH QUESTION: Do multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes? DESIGN: Patients undergoing IVF with homologous single embryo transfer, and who underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy (PGT-A) between 2013 and 2017, were divided into three groups based on degree of embryonic micromanipulation: once-biopsied, once-cryopreserved (group BC, n = 2603), once-biopsied, twice-cryopreserved (group CBC, n = 95) and twice-biopsied, twice-cryopreserved (group BCBC, n = 15). The primary outcome was live birth; secondary outcomes included positive serum pregnancy test, clinical pregnancy and miscarriage. RESULTS: Group CBC had a significantly lower chance of live birth (adjusted RR 0.57, 95% CI 0.41 to 0.79) and clinical pregnancy (adjusted RR 0.67, 95% CI 0.53 to 0.85) compared with group BC. Miscarriage rates were similar between groups BC and CBC (adjusted RR 1.3, 95% CI 0.64 to 2.7). CONCLUSIONS: Multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes. Although PGT-A is thought to improve reproductive outcomes on a per transfer basis, caution must be exercised in counselling patients on the possibility of diminishing returns owing to further embryonic micromanipulation after an embryo has been cryopreserved.

A multi-centre international study of salivary hormone oestradiol and progesterone measurements in ART monitoring.

RESEARCH QUESTION: Ovarian stimulation during IVF cycles involves close monitoring of oestradiol, progesterone and ultrasound measurements of follicle growth. In contrast to blood draws, sampling saliva is less invasive. Here, a blind validation is presented of a novel saliva-based oestradiol and progesterone assay carried out in samples collected in independent IVF clinics. DESIGN: Concurrent serum and saliva samples were collected from 324 patients at six large independent IVF laboratories. Saliva samples were frozen and run blinded. A further 18 patients had samples collected more frequently around the time of HCG trigger. Saliva samples were analysed using an immunoassay developed with Salimetrics LLC. RESULTS: In total, 652 pairs of saliva and serum oestradiol were evaluated, with correlation coefficients ranging from 0.68 to 0.91. In the European clinics, a further 237 of saliva and serum progesterone samples were evaluated; however, the correlations were generally poorer, ranging from -0.02 to 0.22. In the patients collected more frequently, five out of 18 patients (27.8%) showed an immediate decrease in oestradiol after trigger. When progesterone samples were assessed after trigger, eight out of 18 (44.4%) showed a continued rise. CONCLUSIONS: Salivary oestradiol hormone testing correlates well to serum-based assessment, whereas progesterone values, around the time of trigger, are not consistent from patient to patient.

Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.

BACKGROUND: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media. STUDY DESIGN: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo. CONCLUSION: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.

RNA-seq as a tool for evaluating human embryo competence.

The majority of embryos created through in vitro fertilization (IVF) do not implant. It seems plausible that rates of implantation would improve if we had a better understanding of molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer uses an ad hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing (RNA-seq) of embryos could yield high-dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Recent advances enabling the production of RNA-seq libraries from single cells have facilitated the application of this technique to the study of transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to embryo selection procedures by generating RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for assessing embryo competence. Specifically, we show the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype and to identify candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF.

To test or not to test? A framework for counselling patients on preimplantation genetic testing for aneuploidy (PGT-A).

STUDY QUESTION: What is the treatment path and cumulative live birth (CLB) rate from a single oocyte retrieval of patients who intend to pursue PGT-A at the start of an IVF cycle compared to matched controls? SUMMARY ANSWER: The choice of PGT-A at the start of the first IVF cycle decreases the CLB per oocyte retrieval for patients <38 years of age, however patients ≥38 years of age benefit significantly per embryo transfer (ET) when live birth (LB) is evaluated. WHAT IS KNOWN ALREADY: PGT-A has been shown to reduce the practice of transferring multiple embryos and to confer a higher live birth rate per transfer. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study from December 2014 to September 2016, involving 600 patients: those intending PGT-A for their first IVF cycle (N = 300) and their matched controls. Post-hoc power calculations (alpha of 0.05, power of 0.80) indicated that our study was powered adequately to demonstrate significant differences in CLB per retrieval and LB per transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was performed at a large academically affiliated infertility practice where approximately 80% of patients have insurance coverage for fertility care. Patients were identified through electronic medical records, and those who intended to pursue PGT-A at the start of stimulation were assessed. Patients were matched by age, time of oocyte retrieval and oocyte yield to the same number of controls. CLB outcomes per single retrieval, including the fresh and frozen transfers arising from the initial stimulation cycle, were calculated. MAIN RESULTS AND THE ROLE OF CHANCE: PGT-A was not beneficial when CLB rate was assessed per retrieval, however its benefits were significant when LB rate was assessed per transfer. First cycle, <38 year-old patients who intended to have PGT-A had a significantly (P < 0.001) lower CLB rate per oocyte retrieval compared to controls (49.4% vs. 69.1%). Conversely, patients ≥ 38 years in the PGT-A group had similar CLB rates compared to controls per oocyte retrieval, while LB rates per transfer were doubled compared to controls (62.1% vs. 31.7%; P < 0.001). Of the first-cycle PGT-A and control patients, 25.3% and 2.3% failed to achieve a transfer, respectively. LIMITATIONS, REASONS FOR CAUTION: This is not a true intention-to-treat study, due to its retrospective nature. Additionally, the number of patients with two or more previous miscarriages was significantly greater in the PGT-A group as compared to controls, however a sub-analysis showed that this failed to impact outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The findings indicate that PGT-A may be detrimental for those <38 years old undergoing their first IVF cycle. PGT-A has the greatest clinical impact when a transfer is achieved in the ≥38 years old population. This study evaluates the typical treatment path following a patient's choice to pursue PGT-A at the cycle start, and can be used as a guide for counselling patients in relation to age and cycle number. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Hippo signaling in the ovary and polycystic ovarian syndrome.

PURPOSE: To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS. METHODS: We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS. RESULTS AND CONCLUSIONS: Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.

Elevated progesterone and its impact on birth weight after fresh embryo transfers.

PURPOSE: The purpose of the study was to examine the association between serum progesterone levels on the day of hCG administration and birth weight among singleton live births after fresh embryo transfer. METHODS: This study was conducted as a retrospective cohort database analysis on patients who underwent IVF treatment cycles from January 2004 to April 2012. The study was performed at a University affiliated private infertility practice. All cycles that had achieved a singleton live birth after fresh embryo transfer and for which progesterone was measured on the day of hCG administration were examined. Generalized linear models were used to calculate mean birth weight and z-scores. RESULTS: We analyzed 817 fresh IVF embryo transfers in which birth weight, gestational age, and progesterone (ng/mL) level on day of hCG administration were documented. While there was a decrease in birth weight as progesterone quartile [≤0.54; >0.54 to ≤0.81; >0.81 to ≤1.17; >1.17 ng/mL] increased, the difference in mean birth weights among the four quartiles was not statistically significant (p = 0.11) after adjusting for maternal age and peak estradiol levels. When dichotomizing based on a serum progesterone considered clinically elevated, cycles with progesterone >2.0 ng/mL had a significantly lower mean singleton birth weight (2860 g (95% CI 2642 g, 3079 g)) compared to cycles with progesterone ≤2.0 ng/mL (3167 g (95% CI 3122 g, 3211 g) p = 0.007)) after adjusting for maternal age and estradiol. CONCLUSION: We demonstrated that caution should be exercised when performing fresh embryo transfers with elevated progesterone levels and in particular with levels (>2.0 ng/mL) as this may lead to lower birth weight.