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Study question Is leukocyte telomere length (LTL) correlated with cumulus cells telomere length (CCTL) in an age-heterogeneous women population? Summary answer LTL showed a positive correlation with CCTL in the studied population. Hence, its potential value as indicator of ovarian age would deserve further evaluation. What is known already Progressive telomere shortening has been related to ovarian aging and genomic instability during early development. A positive correlation between short telomere length of the first polar body and aneuploidy rate has been reported. CCTL has shown to be a biomarker of oocyte and embryo quality, but its assessment is impractical. LTL has been proposed as a surrogate of TL of follicular cells, but telomere lengthening through folliculogenesis could be controlled by different mechanisms. Thus, we aimed to determine if LTL in an age-heterogeneous population is correlated with CCTL and therefore considered an accurate surrogate for telomere length in the ovary. Study design, size, duration In this prospective non-interventional cohort study, 35 egg donors and 17 women undergoing Preimplantation Genetic Testing for Aneuploidy (PGT-A) treatment were included during sixteen months. Following controlled ovarian stimulation determined by treating physicians, oocyte retrieval was performed 36 hours after final maturation induction. Cumulus cells (CC) for telomere length (TL) measurement were obtained after the pick-up and oocyte stripping. A blood sample was collected through peripheral venous access for LTL measurement. Participants/materials, setting, methods Genomic DNA of CC and leukocytes from the 52 subjects was isolated. Average delta cycle threshold (ΔCt) was determined using a SYBR green quantitative real-time PCR protocol for relative TL. For normalization of measurements, a Taqman assay for the multicopy gene Alu was performed. ΔCtL and ΔCtCC were compared by a paired t-test analysis and the fold change was calculated. Additionally, the association between them and patient age was analyzed by a Pearson correlation test. Main results and the role of chance Mean participant’s age was 29.94 ± 7.55 years and mean values for ΔCtL and ΔCtCC were 7.99 ± 0.53 and 7.46 ± 0.75, respectively. A positive significant correlation was found between age and ΔCt (ΔCtL: R2=0.71, p-value=5.18e–09; ΔCtCC: R2=0.47, p-value=0.00049). Since ΔCt values are inversely proportional to the amount of nucleic acids amplified and, therefore, to the telomere length, this correlation means that TL in both cell types decreases as women age. Additionally, ΔCtL was significantly higher than ΔCtCC (ΔCt fold change: 0.93, p-value=9e–07), meaning that CC showed significantly longer telomeres than leukocytes, thus supporting our previous published results in young egg donors. When analyzing the ΔCtL and ΔCtCC in these age-heterogeneous sample, a positive moderate and significant correlation was observed (R2=0.42, p-value=0.002). Thus, LTL could be suggested as a potential indicator of CCTL and therefore as a candidate for a biological marker of ovarian aging. Limitations, reasons for caution The sample size of this study was moderate and perhaps increasing the number of subjects might give additional strength to our findings. In addition, although relative telomere length allowed for adequate comparison between subjects, this method did not allow for absolute TL measurement. Wider implications of the findings: While reproductive implications of LTL measurement need to be further studied, our results support the potential usefulness of LTL measurement as an indicator of CCTL and ovarian aging when analyzing an age-heterogeneous population. Further, our findings suggest that CC could possess different mechanisms to cope against telomere length shortening. Trial registration number Not applicable

Mitochondria play an essential role in generating energy for embryo development and maintaining embryo metabolism through key cellular functions including ion homeostasis, amino acid metabolism, glycolysis, fatty acid metabolism, signal transduction and apoptotic regulation. Recent literature suggests that mitochondrial content and function may be related to implantation success and embryo viability. Some studies have linked increased levels of mitochondrial DNA to aneuploidy, advanced maternal age and euploid blastocyst with implantation failure, while others have failed to demonstrate similar findings. This review aims to provide an overview of the current literature surrounding the possibilities of using mitochondria as an additional biomarker for infertility treatment outcome and summarize the reasons as to why there are inconsistencies in these studies.

To examine the existing literature in regards to the relationship between assisted reproductive technologies (ART) and low birth weight (LBW).In 2017, Martin et al. reported on the incidence of low birth weight in relation to the number of embryos transferred, and showed that incidence of low birth weight in singletons correlates with number of embryos transferred. Meanwhile, several studies have shown increased weight of singletons born after frozen embryo transfers compared with fresh embryo transfers. A recent study published by Sekhon et al., among others, disputes these findings, and claims that frozen and fresh embryo transfers result in comparable birth weights. It is also noteworthy that Mass et al., in 2016, analyzed how birth weight as a result of assisted reproductive technologies has evolved over the years, and concluded that birth weight has not changed significantly over a long period of time.Newborns conceived via assisted reproductive technologies are three times more likely to have low birth weight. Although multiple gestation and its associated prematurity are the main risk factors for low birth weight in ART-conceived pregnancies, some of the other processes specific to assisted reproduction also impact perinatal outcomes. Options, such as fresh or frozen embryo transfers, the number of embryos transferred, or endometrial preparation may all importantly affect birth weight and prematurity of ART-conceived newborns.

Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3′-untranslated region (3′-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process.

Mitochondria play a central role in the regulation of energy metabolism in oocytes and preimplantation embryos, where the number and morphology of mitochondria and mitochondrial DNA (mtDNA) content are tightly regulated. A number of mouse models with mitochondrial dysfunction result in infertility, further confirming the key role of mitochondria in female reproductive function. When cells and organisms detect mitochondrial dysfunction they use response mechanisms directed at recovering salvageable mitochondria and eliminating mitochondria that can no longer be rescued. Among these mechanisms, mitochondrial unfolded protein response (UPRmt) has recently been linked with prevention of aging, as compromised mitochondrial stress response contributes to age-related accumulation of damaged proteins, reduced oxidative phosphorylation, and increased reactive oxygen species (ROS) production. These mechanisms seem to be especially relevant for reproduction, as targeted deletion of the UPRmt–regulatory gene Clpp results in female infertility, with impaired oocyte maturation and two-cell embryo development, and failure to form blastocysts. In addition, absence of CLPP results in accelerated depletion of follicles, and a phenotype similar to premature reproductive aging. Further studies will provide novel mechanistic insights for physiologic and pathologic control of oocyte and early embryonic mitochondrial function, which can be exploited for the development of novel therapeutic approaches for the promotion of fertility during the aging process.

Objective To determine whether increased endometrial B-cell lymphoma 6 (BCL6) expression is associated with live birth in a normal responder in vitro fertilization (IVF) population. Design Case-control study. Setting University-affiliated infertility center. Patient(s) Two groups of women undergoing IVF with preimplantation genetic testing for aneuploidy followed by warmed, single, euploid embryo transfer. Group 1 consisted of women who failed to achieve live birth, and group 2 consisted of women who achieved live birth. Intervention(s) None. Main Outcome Measure(s) Endometrial BCL6 expression measured by immunohistochemistry in endometrial tissue samples. Overexpression was defined by mean HSCORE with a cutoff of positivity of >1.4, as previously described in the literature. Result(s) Twenty-seven patients who achieved live birth and 23 patients who failed to achieve live birth were included. B-cell lymphoma 6 expression/HSCORE and live birth rate were not associated (Odds ratio [OR], 0.78 [0.24–2.55]). Using a cutoff of >1.4 for positivity, 8 of 23 samples were positive for BCL6 in the no live birth group, whereas 7 of 27 were positive in the live birth group. There was no significant association between BCL6 positivity and live birth (OR, 0.66 [0.19–2.21]). Conclusion(s) The proportion of patients with BCL6 positivity did not significantly differ between those who achieved live birth and those who did not. In the population of patients at our center, who compromise of women who respond normally to IVF stimulation, BCL6 overexpression was not associated with IVF success. Physicians implementing BCL6 testing as a diagnostic tool for clinical decision making should counsel patients that results may have limited utility in predicting IVF outcomes in this population.

PURPOSE: To evaluate embryology and pregnancy outcomes following individual and group embryo culture in the setting of contemporary laboratory practices and freeze-all cycles. METHODS: Patients underwent ovarian stimulation followed by intracytoplasmic sperm injection (ICSI). Embryos proceeded through individual culture and then underwent preimplantation genetic testing for aneuploidy (PGT-A) via trophectoderm biopsy. In a subsequent cycle, participants underwent single embryo transfer of a vitrified-warmed, euploid embryo. Outcomes were compared to controls undergoing group culture during the same time frame. The Mann-Whitney U test and logistic regression models were utilized. RESULTS: Outcomes were assessed for 144 patients whose embryos underwent individual culture and 449 controls whose embryos underwent group culture. There were no significant differences in fertilization rates between groups (81.7% for individual culture vs. 84.1% for group culture, p = 0.22). However, individual culture was associated with a decreased rate of blastocyst formation compared to group culture (43.5% vs. 48.5%, p < 0.01). Following single, vitrified-warmed euploid blastocyst transfer, there were no significant differences between individual culture and group culture, respectively, in rates of positive βhCG (81.9% vs. 81.5%, p = 0.91), sustained implantation (63.9% vs. 65.0%, p = 0.80), biochemical miscarriage (16.7% vs. 12.3%, p = 0.18), or clinical miscarriage (1.4% vs. 4.2%, p = 0.13). CONCLUSION: While individual culture appears to negatively impact the rate of usable blastocyst formation compared to group culture, there were no significant differences in pregnancy outcomes following transfer of a single, vitrified-warmed euploid blastocyst.

To investigate whether ovarian fragmentation for follicular activation (OFFA) improves ovarian reserve markers and in vitro fertilization (IVF) outcomes in women with poor ovarian response (POR).Randomized, controlled trial, with parallel assignment.University hospital.Thirty-four women with POR according to the European Society of Human Reproduction and Embryology criteria.Women with POR were randomly allocated to receive ovarian fragmentation in 1 ovary or to no intervention (control group). Ovarian reserve markers were followed at 2-week intervals for 6 months. In vitro fertilization cycles were initiated when the antral follicle count (AFC) doubled or at the end of follow-up.The primary outcome was the number of metaphase II (MII) oocytes obtained. Antral follicle count, antimüllerian hormone level, and reproductive outcomes were recorded as secondary outcomes. Exploratory outcomes included surgical results and analysis of protein and gene expression.Ovarian fragmentation for follicular activation resulted in an increase in AFC in the intervention ovary compared with the control ovary and an increase in total AFC in the OFFA group compared with controls. Serum antimüllerian hormone and follicle-stimulating-hormone levels did not improve in the OFFA group throughout the follow-up period. Fifteen patients from each arm underwent IVF. In the control group, 33 MII oocytes were retrieved and 18 embryo transfers were performed, with a 20% pregnancy rate and an 18.7% live birth rate per cycle. In the OFFA group, 23 MII oocytes were retrieved and 11 embryo transfers were performed, with a 13.3% pregnancy rate and a 6.7% live birth rate per cycle. Reproductive outcomes did not significantly differ between the groups. Hippo pathway inhibition was confirmed by an 18.8% reduction in the phospho-YAP/YAP (Yes-associated protein 1) ratio and BIRC and CCN overexpression after fragmentation.Ovarian fragmentation for follicular activation in women with POR resulted in an increase in AFC but did not modify IVF outcomes when compared with controls.NCT02354963.

PURPOSE OF REVIEW To discuss changes in female demographic parameters in the US and associated increase in utilization of fertility services. RECENT FINDINGS Fractions of women earning bachelor's, master's, and doctoral degrees increased from 1970 to 2018 (32.6 vs 64.8; 7.9 vs 27.3; 0.54 vs 5.7 per 10,000 women; P

To discuss the utilization, performance, and interpretation of noninvasive prenatal testing (NIPT) results in women achieving pregnancy through in vitro fertilization (IVF) and preimplantation genetic testing for aneuploidy (PGT-A).Although PGT-A is a highly accurate method for the selection of euploid embryos the possibility for error still exists. Many women pursue NIPT after conception via IVF with or without PGT-A, whereas some forgo prenatal screening all together. Recent evidence suggests that the prevalence of a positive NIPT following PGT-A is low, and the positive predictive value is altered in this population.NIPT is a valuable prenatal screening tool that should be offered to pregnant women regardless of prior PGT. In women who conceive following IVF and PGT-A through the transfer of euploid embryos, positive test results should be interpreted with caution.

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.

International audience; Objective: To study the true prevalence of recurrent implantation failure.Design: Retrospective cohort study.Setting: A private assisted reproductive technology center.Patient(s): Women (n- 4,429) with anatomically normal uterus who underwent up to three consecutive frozen euploid single embryo transfers (FE-SETs) were included in the study. Cycles with donor eggs or gestational carriers were excluded.Intervention(s): None.Main Outcome Measure(s): Cumulative outcomes from these cycles were analyzed. A logistic regression model was used to assess the differences of outcomes between first, second, and third FE-SET and a Kaplan-Meier curve as used to analyze cumulative implantation rate.Result(s): The mean age of the patients included in the study was of 35.4 years. The sustained implantation rates of the first, second, and third FE-SET were 69.9%, 59.8%, and 60.3% per transfer, respectively. The cumulative sustained implantation rate after up to three consecutive FE-SET was 95.2%. The live birthrates after the first, second, and third FE-SET were 64.8%, 54.4%, and 54.1% per transfer, respectively. The cumulative live birth rate after up to three consecutive FE-SET was 92.6%. The miscarriage rate after observing a positive heartbeat was not different between the first (7.2%), second (8.8%), and third (12.7%) FE-SET.Conclusion(s): Our findings suggest that true recurrent implantation failure is rare. For those patients with the ability to make euploid blastocysts

The formation of differential cell lineages in the mammalian blastocyst from the totipotent zygote is crucial for implantation and the success of the whole pregnancy. The first lineage segregation generates the polarised trophectoderm (TE) tissue, which forms the placenta, and the apolar inner cell mass (ICM), which mainly gives rise to all foetal tissues and also the yolk sac1–3. The mechanism underlying this cell fate segregation has been extensively studied in the mouse embryo4,5. However, when and how it takes place in the human embryo remains unclear. Here, using time-lapse imaging and 325 surplus human embryos, we provide a detailed characterisation of morphological events and transcription factor expression and localisation to understand how they lead to the first lineage segregation in human embryogenesis. We show that the first lineage segregation of the human embryo is triggered by cell polarisation that occurs at the 8-cell stage in two sequential steps. In the first step, F-actin becomes apically polarised concomitantly with embryo compaction. In the second step, the Par complex becomes polarised to form the apical cellular domain. Mechanistically, we show that activation of Phospholipase C (PLC) triggers actin polarisation and is therefore essential for apical domain formation, as is the case in mouse embryos6. Finally, we show that, in contrast to the mouse embryo, the key extra-embryonic determinant GATA37,8 is expressed not only in extra-embryonic lineage precursors upon blastocyst formation. However, the cell polarity machinery enhances the expression and nuclear accumulation of GATA3. In summary, our results demonstrate for the first time that cell polarisation reinforces the first lineage segregation in the human embryo.

Objective To investigate the impact of obesity as determined by bioelectric impedance analysis (BIA) and body mass index (BMI) on in vitro fertilization (IVF) laboratory and clinical outcomes. Design Prospective cohort study. Setting Academic-affiliated private practice. Patient(s) A total of 1,889 infertile couples undergoing IVF from June 2016 to January 2019. Intervention(s) Female patients and male partners underwent BIA and BMI measurement at the time of oocyte retrieval. Embryology and clinical outcomes were prospectively tracked with comparison groups determined by percentage of body fat (%BF) and BMI categories. Main Outcome Measure(s) Fertilization rate, blastocyst formation rate, euploidy rate, miscarriage rate, sustained implantation rate, live birth rate, rates of low birth weight/very low birth weight, prematurity rates. Result(s) Fertilization rates and euploidy rates were equivalent among all women. Blastocyst formation rates were slightly higher (55%) in women with an obese %BF compared with all other %BF categories (51%); however, this trend was not noted in women defined as obese by BMI. Miscarriage rates, sustained implantation rates, and live birth rates were equivalent among all women. The rate of very low birth weight was low but increased in obese women (3%) versus underweight, normal-weight, and overweight counterparts (0%–1.3%) as determined by %BF and BMI. Obesity in men did not significantly affect any embryologic or clinical outcomes. Conclusion(s) Although maternal obesity imposes a small but increased risk of very low birth weight infants, most embryology and pregnancy outcomes are equivalent to normal weight patients. Paternal obesity does not appear to affect IVF, pregnancy, or delivery outcomes.

To evaluate whether the telomere length of white blood cells (WBC) and cumulus cells (CC) in an infertile population is associated with ovarian and embryonic performance.Prospective cohort study.Academic-affiliated private practice.A total of 175 infertile women undergoing in vitro fertilization (IVF) at a single center between July 2017 and December 2018.On the day of oocyte retrieval, genomic DNA was isolated from WBC and CC samples. Telomere length assessment was performed for both tissue types using quantitative real-time polymerase chain reaction. Telomere lengths were normalized using an AluYa5 sequence as an endogenous control, and linear regressions were applied.This study assessed the relationship between relative telomere length of WBC and CC samples and measures of ovarian and embryonic performance. Specifically, patient age, antimüllerian hormone (AMH) level, peak estradiol (EThere was a statistically significant relationship between WBC relative telomere length and patient age as well as rates of embryonic aneuploidy, with shorter WBC relative telomere length associated with increasing patient age (P.01) and higher rates of aneuploidy (P=.01). No statistically significant relationships were observed between WBC relative telomere length and the other outcome measures. No significant associations were noted between CC relative telomere length and any outcomes assessed in this study.The relationship between WBC relative telomere length and aneuploidy warrants further investigation, particularly because significant overlap exists between increasing maternal age and rates of embryonic aneuploidy.

PURPOSE: As obesity becomes increasingly prevalent, its impact on fertility has been a subject of great debate. Nearly all prior research is retrospective and evaluates obesity utilizing body mass index (BMI), which may overestimate adiposity in individuals with a greater amount of lean muscle and underestimate adiposity in those with less muscle mass. METHODS: We prospectively evaluated 2013 couples undergoing infertility treatment with in vitro fertilization (IVF). Percent body fat (%BF) was measured by use of a bioelectric impedance analysis (BIA) scale at baseline. BMI was also determined. Ovarian reserve parameters, ovarian response to controlled ovarian hyperstimulation, and semen analyses were measured in correlation with their %BF and BMI. RESULTS: Females classified as obese based on %BF or BMI had lower serum FSH. However, when the analysis was limited to women without PCOS (n = 1706), obesity based on %BF or BMI was associated with lower serum AMH. Female obesity—regardless of a PCOS diagnosis—did not affect number of mature oocytes retrieved. Males who were in obese %BF category were found to have lower TMSC compared with normal weight counterparts (p < 0.05); however, the observed decrease was not significant enough to limit the success of assisted reproductive technologies. CONCLUSIONS: These findings suggest that while obesity may affect ovarian reserve in women variably depending on presence of PCOS, it does not affect number of mature oocytes available after COH. Similarly, while a high %BF in males is associated with lower TMSC, the observed difference is unlikely to affect IVF 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

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 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.

PURPOSE: The rate of embryonic aneuploidy increases with increasing female age and is the primary cause of lower pregnancy and live birth rates (LBR) in older reproductive age women. This retrospective cohort study evaluates single euploid embryo transfers to determine whether an age-related decline in reproductive efficiency persists. METHODS: A total of 8175 non-donor single embryo transfers (SET) after pre-implantation testing for aneuploidy (PGT-A) and cryopreservation were included. These were divided into five groups by patient age: 42 (n = 243). Implantation rate (IR), clinical pregnancy rate (CPR), and LBR were calculated for each group as a percentage of embryos transferred and compared. CPR was also analyzed as a percentage of implanted pregnancies, and LBR as a percentage of clinical pregnancies, to determine when age has the greatest impact. These results were then adjusted for confounding variables via a multivariate logistic regression model. RESULTS: Implantation rates negatively correlated with age. After adjusting for confounders, women 38 years or older had a significantly lower IR than those under 35 (OR 0.85, 95%CI 0.73–0.99 for 38–40 years old; 0.69, 0.53–0.91 for 41–42, and 0.69, 0.51–0.94 for > 42). These differences are also apparent in CPR and LBR. The rates of progression to clinical pregnancy and live birth did not differ significantly by age group. Other factors observed to affect IR independently were anti-Müllerian hormone (AMH), day of embryo transfer, and embryo morphology. CONCLUSION: While selection of euploid embryos may be effective in overcoming a significant proportion of the age-related decline in reproductive efficiency, a decrease in IR, CPR, and LBR persists even when analyzing only euploid embryo transfers. The observed impact of aging is, therefore, independent of ploidy, as well as of other variables that affect reproductive efficiency. These results indicate that factors other than aneuploidy contribute to reproductive senescence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01739-0) contains supplementary material, which is available to authorized users.

STUDY QUESTION Do supraphysiologic estradiol (E2) levels in the ranges attained during normal and high response superovulation cycles modify the onset of endometrial secretory transformation? SUMMARY ANSWER Highly supraphysiologic levels of E2 do not alter the ability of physiologic levels of progesterone (P4) to induce secretory transformation. WHAT IS KNOWN ALREADY Previous studies have demonstrated that premature P4 elevations during IVF cycles are associated with a decrement in clinical pregnancy rates after fresh embryo transfer due to shifts in the window of implantation (WOI). However, alterations in the onset of secretory transformation may not apply uniformly to all patients. High responders with supraphysiologic E2 levels accompanied by similar subtle increases in P4 have not been shown to have decreased sustained implantation rates. This prospective investigation in which whole-genome transcriptomic and methylomic analysis of the endometrium is performed for individual patients under a range of E2 concentrations brings clarity to a long-debated issue. STUDY DESIGN, SIZE, DURATION A randomized, prospective and paired trial was conducted in which 10 participants were enrolled and randomized to the order in which they completed three distinct uterine stimulation cycles, each at a specific E2 concentration: physiologic (∼180 pg/ml), moderately supraphysiologic (600–800 pg/ml) or supraphysiologic (2000 pg/ml). Target E2 ranges were selected to mimic those seen in natural, controlled ovarian stimulation and IVF cycles. E2 valerate was administered in order to maintain stable E2 levels for 12 days followed by intramuscular P4 in oil 10 mg/day for two doses, after which an endometrial biopsy was performed. A total of 30 endometrial biopsies were included in a whole-genome transcriptomic and methylomic analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Healthy volunteers without a history of infertility were included in this study at a single large infertility center. DNA was isolated from the endometrial biopsy specimens and bisulfite sequencing was performed to construct a methylation array. Differential methylation analysis was conducted based on differences in M-values of individuals across treatment groups for each probe as well as carrying out t-tests. RNA was isolated for RNA-Seq analysis and gene expression values were compared using DESeq2. All analyses were performed in a pairwise fashion to compare among the three stimulation cycles within individuals and secondarily to compare all participants in each of the cycles. MAIN RESULTS AND THE ROLE OF CHANCE The mean peak E2 and P4 levels were 275 pg/ml and 4.17 ng/ml in the physiologic group, 910 pg/ml and 2.69 ng/ml in the moderate group was, and 2043 pg/ml and 2.64 ng/ml in the supraphysiologic group, respectively. Principal component analysis of 834 913 CpG sites was performed on M-values of individuals within the low, moderate and supraphysiologic conditions in a paired approach. There were no differences in genome-wide methylation within participants across E2 groups. A paired analysis revealed that gene expression profiles did not differ within the same individual at each of the three E2 levels. No significant alterations in gene expression as related to endometrial physiology were identified between the low, moderate and supraphysiologic groups in an inter-participant analysis. LIMITATIONS, REASONS FOR CAUTION Although each participant completed a physiologic cycle in which E2 levels were maintained in a range that would simulate a natural cycle, our findings are limited by lack of an unmedicated control to assess if there was a potential effect from E2V. Additionally, our results were obtained in fertile individuals, who may have a different endometrial response compared to an infertile population. Despite the whole genomic endometrial assessment and rigorous, paired study design, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS Given that the endometrial response to P4 is unaffected by E2 levels in the supraphysiologic range, diminutions in implantation seen in stimulated cycles may result from embryonic-endometrial dyssynchrony following early P4 elevations or slowly blastulating embryos, which occur independently of the magnitude of the E2 rise. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. Dr E.S. reports consultancy work for The Foundation for Embryonic Competence, Basking Ridge, NJ, USA. The other authors declare no conflict of interests related to this topic. TRIAL REGISTRATION NUMBER NCT02458404.

To provide an overview of mitochondrial functional alterations in women with polycystic ovary syndrome (PCOS).Although numerous studies have focused on PCOS, the pathophysiological mechanisms that cause this common disease remain unclear. Mitochondria play a central role in energy production, and mitochondrial dysfunction may underlie several abnormalities observed in women with PCOS. Recent studies associated mtDNA mutations and low mtDNA copy number with PCOS, and set out to characterize the potential protective role of mitochondrial and endoplasmic reticulum unfolded protein responses (UPR and UPR).Mitochondrial dysfunction likely plays a role in the pathogenesis of PCOS by increasing reactive oxygen (ROS) and oxidative stress. This occurs in a metabolic milieu often affected by insulin resistance, which is a common finding in women with PCOS, especially in those who are overweight or obese. Mutations in mtDNA and low mtDNA copy number are found in these patients and may have potential as diagnostic modalities for specific PCOS phenotypes. More recently, UPR and UPR are being investigated as potential cellular rescue mechanisms in PCOS, the failure of which may lead to apoptosis, and contribute to decreased reproductive potential.

Objective To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. Design Prospective cohort study. Setting Not applicable. Patients A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018. Interventions None. Main Outcome Measures All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the β-actin were used. Data were statistically analyzed by analysis of variance with the Scheffe multiple pairwise comparison for categorical variables and by linear regression for numerical variables. Results Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. Conclusions During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

As women delay childbearing because of demographic and socioeconomic trends, reproductive aging and ensuing ovarian dysfunction become increasingly more prevalent causes of infertility. Age-related decline in fertility is characterized by both quantitative and qualitative deterioration of the ovarian reserve. Importantly, disorders of aging are frequently associated with mitochondrial dysfunction, as are impaired oogenesis and embryogenesis. Ongoing research explores the role of mitochondrial dysfunction in ovarian aging, and potential ways to exploit mitochondrial mechanisms to slow down or reverse age-related changes in female gonads.

OBJECTIVE: To determine the true prevalence of recurrent implantation failure (RIF). DESIGN Retrospective cohort study METHODS: Women (n=4,429) with anatomically normal uterus who underwent up to three consecutive euploid frozen SETs were included in the study. Cycles with donor eggs or gestational carriers were excluded. Cumulative outcomes from these cycles were analyzed. A logistic regression model was employed to assess the differences of outcomes between first, second, and third euploid SET and a Kaplan-Meier curve as utilized to analyze cumulative implantation rate. RESULTS: The mean age of the patients included in the study was of 35.4±4.2 years. The sustained implantation rates (SIR) of the 1 st , 2 nd and 3 rd FE-SET were 69.9%, 59.8%, and 60.3% per transfer, respectively. The cumulative SIR after up to 3 consecutive FE-SET was 95.2% (95% CI: 94.0%-96.2%). The live birth rates (LBRs) after the 1 st , 2 nd and 3 rd FE-SET were 64.8%, 54.4%, and 54.1% per transfer, respectively. The cumulative LBR after up to 3 consecutive FE-SET was 92.6% (95% CI: 91.2%-93.9%). The miscarriage rate after observing a positive heartbeat was not different between the 1 st (7.2%, 95%CI: 6.4%-8.2%), 2 nd (8.8%, 95% CI:6.3%-11.7%) and 3 rd (12.7%, 95%CI: 6.2%-22.0%) FE-SET. CONCLUSIONS: Our findings suggest that true RIF is rare. For those patients with the ability to make euploid blastocysts, less than 5% would fail to achieve a clinical pregnancy with 3 embryos transferred. It remains to be further investigated whether this threshold identifies a truly recalcitrant group, or simply a statistical certainty based on random variation. FUNDING STATEMENT: All funding was internal from one program – RMA New Jersey, Basking Ridge, US. No outside funding was provided, thus no commercial interest for anyone involved. RMA New Jersey does not collect any money for the performance of PGT-A and thus does not resale or profit from it. Patients were not charged for PGT-A screening. DECLARATION OF INTERESTS: E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence; he is co-founder and a shareholder of ACIS. All other authors have no competing interests to disclose. ETHICS APPROVAL STATEMENT: No embryos were biopsied specifically for the purpose of this study. Given the retrospective nature of this study, the access and processing of patient data was approved by the Advarra Institutional Review Board under a protocol for retrospective studies - protocol number Pro00027158.

While DNA provides all the necessary information for normal cell development, the expression of specific genes varies in different body tissues and transcriptional program is determined by cell fate and function. The differences in the interpretation of DNA by different cell types within the same organism are largely mediated by epigenetic mechanisms. Epigenetic marks help determine how DNA is structurally organized and which sections are available for transcription, without causing a change in the DNA sequence. DNA methylation, histone modifications, and noncoding RNAs are major players of the cellular epigenetic machinery in mammals. Epigenetic changes are remarkably associated with reproductive aging in both males and females. They are also responsible for the phenomenon of genomic imprinting. This chapter highlights the most significant molecular features of epigenetic regulatory mechanisms and how they can be studied. Furthermore, special emphasis is given on how and when major epigenetic events during embryo development overlap with assisted reproductive technology interventions and potential consequences. Moreover, this chapter also provides recent examples of how the epigenome can be exploited as a tool to improve clinical outcomes in assisted reproductive treatment.

An algorithm assessing the methylation levels of 353 informative CpG sites in the human genome permits accurate prediction of the chronologic age of a subject. Interestingly, when there is discrepancy between the predicted age and chronologic age (age acceleration or “AgeAccel”), patients are at risk for morbidity and mortality. Identification of infertile patients at risk for accelerated reproductive senescence may permit preventative action. This study aimed to assess the accuracy of the “epigenetic clock” concept in reproductive age women undergoing fertility treatment by applying the age prediction algorithm in peripheral (white blood cells [WBCs]) and follicular somatic cells (cumulus cells [CCs]), and to identify whether women with premature reproductive aging (diminished ovarian reserve) were at risk of AgeAccel in their age prediction. Results indicated that the epigenetic algorithm accurately predicts age when applied to WBCs but not to CCs. The age prediction of CCs was substantially younger than chronologic age regardless of the patient’s age or response to stimulation. In addition, telomeres of CCs were significantly longer than that of WBCs. Our findings suggest that CCs do not demonstrate changes in methylome-predicted age or telomere-length in association with increasing female age or ovarian response to stimulation.

STUDY QUESTION Do infertile women aged

Purpose of review To discuss the common aspects of energy generation in the cell, substrate utilization by the oocyte and the preimplantation embryos, metabolic characteristics of the reproductive tract, and how metabolic characteristics of an embryo can be exploited to assess viability. Recent findings Utilization of pyruvate, lactate and glucose by the oocyte and the preimplantation embryo seems to be tightly controlled by the most essential molecular regulatory pathways. Summary Accumulating data on the metabolic needs of the preimplantation embryo and the substrate concentrations in the reproductive tract guide us in improving in-vitro culture conditions, and provide potential biomarkers of embryo viability.