This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52

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 mu l 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).

Research question: What areas of manual IVF cryostorage operations are common to the safe operation of IVF cryostorage facilities and require effort from embryologists? Design: Observational time and motion data were collected by two observers equipped with the digital cameras over 2 weeks at four well-characterized US IVF centres (sites a, $, g and d) from 12 participants performing cryostorage tasks. To understand the work processes of the different sites and assist in the data analysis, informal interviews were conducted with the study participants and laboratory directors. Data were analysed to identify work processes that might be eliminated or diminished by automation and software improvements. Results: On average, it took 3.4 data record queries per retrieval from cryostorage to identify a cane, while the canister was lifted an average of 1.5 times per retrieval, with a mean 11.8 9.2 s per lift. Of the total time spent working with cryostorage equipment, 47.25% was of a fatiguing nature. Sites a, $ and g utilized one person to fi ll the liquid nitrogen storage Dewars, while site d had two technicians working in tandem to move and fi ll the Dewars, with different frequencies and determination factors for refills fi lls and efficiencies. fi ciencies. Conclusions: This time and motion study demonstrated significant fi cant time investment, task redundancy and fatiguing working conditions among embryologists using manual cryostorage processes. There was a disparity of processes and space capacity across different laboratories. Some of these issues may be addressed by the integration of automation and technology solutions.

Single cell analysis of embryos has its limitations, since the specific positioning of cells within the embryo is unknown. Our aim was to use a novel in situ gene expression technology to analyze the expression of up to 80 genes per cell in the human blastocyst and three-dimensionally map these genes within each cell to gain a better understanding of their specific location. We performed targeted in situ RNA sequencing of 80 genes in 15 human blastocysts using sequential rounds of RNA in situ sequencing. Specific RNA transcripts were hybridized then amplified for the chosen genes. The read out was then done over several rounds of in situ sequencing. With each round, the transcript was labelled, and the entire volume of the embryo was imaged. This technique was first performed on mouse blastocysts and the spatial distribution of ∼150 genes was charted in mouse embryos using publicly available single cell RNAseq data sets of preimplantation mouse embryos. Similarly, the publicly available RNAseq data of human blastocysts was used to construct similar blastocysts lineage markers. We performed targeted in situ RNA sequencing of 80 genes in 15 human blastocysts and distinguished three distinct cell types: trophectoderm (TE), Epiblast (Epi), and Primitive Endoderm (PrE). Using the high multiplexing capacity of the 80-gene panel, we identified subpopulations and specific positioning of Epi and TE, including polar TE (pTE) and mural TE (mTE), and discovered novel gene markers expressed explicitly in these populations. By investigating the gene expression patterns within these subpopulations, we identified gene regulatory programs specific to the Epi and each TE subtype, providing deeper insights into the molecular mechanisms underlying Epi and TE differentiation and development in human blastocysts. In addition, we compared our findings in human blastocysts to mouse embryos. Through this comparison, we assessed cell type programs' expression and spatial localization and identified conserved and divergent gene expression patterns between human and mouse embryos. Furthermore, we compared the gene regulatory modules in polar and mural TE and found differences in the expression of genes related to cell adhesion and differentiation. Our study demonstrates the power of targeted in situ RNA sequencing to identify and localize the expression of multiple genes simultaneously within the human blastocyst. By using a high multiplexing capacity of 80 genes, we were able to identify subpopulations of Epi and TE and define gene regulatory programs specific to each subtype. Our results provide a comprehensive view of gene expression dynamics during early human development and could aid in the understanding of the molecular mechanisms underlying Epi and TE differentiation and development. Overall, our approach could be applied to other tissues and developmental stages, providing a valuable resource for future studies on gene expression during development and disease.

Maternal age has a significant impact on the outcome of in vitro fertilization (IVF). However, it is important to delineate outcomes in relation to the contribution of the oocyte and uterus in relation to age. Although age related oocyte chromosomal factors are clearly detrimental, the role of the uterus in older patients is not as clear [1, 2]. The objective of this study was to evaluate IVF outcomes in patients over the age of 50 undergoing donor oocyte cycles. This was a retrospective cohort study that included all embryo transfer cycles using donor oocytes between 01/2015-12/2021 in patients >40 years of age. Data on BMI, paternal age, type of cycle (natural vs. medicated), number of embryos transferred, was evaluated in relation to miscarriages and live births when comparing age groups of 40-44, 45-49 and >50. The statistical analysis was performed using a logistic regression model. A total of 2,087 embryo transfer cycles using donor oocytes in patients >40 years were performed during the study period, of which 1,207 were in patients 40-44, 749 in patients 45-49 and 131 in patients >50 years of age. The mean number of embryos transferred was 1.21±0.41. The presence of implantation was significantly lower in patients >50 (P=0.011), and the presence of a live birth was significantly lower in patients 45-49 and >50, compared to those 40-44 (P=0.018 and P=0.014, respectively). This relationship persisted after adjusting for BMI, paternal age, type of embryo transfer cycle (natural cycle vs medicated cycle) and type of oocyte donor (fresh vs frozen oocyte donor) (Table 1). While oocyte quality is a major determinant of IVF outcomes, additional factors such as uterine aging must also be considered. Even with the use of donor oocytes, maternal age appears to have an impact on implantation and subsequent live birth.

Metabolic function changes through meiosis and is critical for the oocyte developmental competence. As NADH and FAD+ are both autofluorescent, monitoring their fluorescence yields valuable information for characterizing cellular metabolism. Fluorescence Lifetime Imaging Microscopy (FLIM) generates timelapse profiling of metabolic state, without the need for additional markers. Superior to intensity-only measurements, FLIM generates 3 additional parameters (fraction bound, long and short lifetime) that are sensitive to metabolic shifts. In this study, we performed metabolic imaging on GVs and compared the data between oocytes that matured in vitro versus oocytes that did not mature. Additionally, we examined metabolic data of oocytes from young vs advanced maternal age (AMA) women.

To examine the efficacy of a telehealth based approach to effective weight management interventions in women with obesity seeking treatment for infertility.

Research question: Can we develop an interpretable machine learning model that optimizes starting gonadotrophin dose selection in terms of mature oocytes (metaphase II [MII]), fertilized oocytes (2 pronuclear [2PN]) and usable blastocysts?Design: This was a retrospective study of patients undergoing autologous IVF cycles from 2014 to 2020 (n = 18,591) in three assisted reproductive technology centres in the USA. For each patient cycle, an individual dose-response curve was generated from the 100 most similar patients identified using a K-nearest neighbours model. Patients were labelled as dose-responsive if their dose-response curve showed a region that maximized MII oocytes, and flat -responsive otherwise.Results: Analysis of the dose-response curves showed that 30% of cycles were dose-responsive and 64% were flat-responsive. After propensity score matching, patients in the dose-responsive group who received an optimal starting dose of FSH had on average 1.5 more MII oocytes, 1.2 more 2PN embryos and 0.6 more usable blastocysts using 10 IU less of starting FSH and 195 IU less of total FSH compared with patients given non-optimal doses. In the flat-responsive group, patients who received a low starting dose of FSH had on average 0.3 more MII oocytes, 0.3 more 2PN embryos and 0.2 more usable blastocysts using 149 IU less of starting FSH and 1375 IU less of total FSH compared with patients with a high starting dose.Conclusions: This study demonstrates retrospectively that using a machine learning model for selecting starting FSH can achieve optimal laboratory outcomes while reducing the amount of starting and total FSH used.

The use of ICSI in non-male factor infertility cases has dramatically increased worldwide and in many clinics is used for all cases, regardless of sperm characteristics. The objective of this study was to examine differences in euploidy rates between CI and ICSI in non-male factor infertility cases.

To observe the amount of time spent in fatiguing work (bending, lifting and usage of stools and ladders) in the management of in vitro fertilization (IVF) biorepositories.

Current methods for cryostorage in IVF clinics require manual retrieval of specimens from Dewars, canisters, canes, and goblets. The methods used for identification and retrieval of specimens are often not well characterized, and areas for improvement have gone unrealized regarding modernizing specimen cryostorage. The study included embryologists from four independent U.S. IVF clinics who were observed conducting clinical duties, under an IRB approved protocol, over an 8-week total period from August to November 2019. Embryologists were observed and video recorded while preforming routine daily tasks. Three measurable parameters were then documented in relation to the retrieval of viable specimens from storage Dewars: 1) the number of times the embryologist referenced the data record; 2) the number of times (pulls) the canister was lifted into the neck of the Dewar; and 3) the total time the canister was held in the neck. For each pull, a qualitative grade of 1 was assigned if the canister remained comfortably low in the neck of the Dewar; 2, if the canister was positioned close to the top of the neck; and 3, if the canister reached the top of the neck or came out of the Dewar. A total of 95 specimen retrievals were evaluated. A mean of 27.8±16.1 specimen retrievals were recorded per site. Subjects referred to the specimen data records 323 times; on average, it took 3.4 data record queries per retrieval to verify the identity of the cane. Of the 95 retrievals, 89 were from Dewars with necks. The canister was lifted an average of 1.5 times per retrieval, with a mean 11.8±9.2 seconds per pull, and a mean grade of 1.4±0.5. The minimum time for the canister remaining in the neck was 2 seconds with 50 seconds as the maximum. This study identified variance between the number of pulls and the time a canister spent in the neck of the Dewar, when retrieving cryopreserved specimens. Additional queries of the data demonstrated an increase in the time required to retrieve specimens. Eliminating the need for repeated manual review of data records, and more than one pull per retrieval, could help mitigate and lessen the opportunity for any mishandling of cryopreserved specimens. Due to the variability observed in this study, further investigation of both temperature fluctuations throughout specimen retrieval and the subsequent biological effect on the specimens is warranted.

Longitudinal evaluation of the psychological impact of the COVID-19 pandemic on pregnant women following assisted reproductive technology (ART) and to compare them to women seeking infertility treatment. Prospective longitudinal cohort study from April to June 2020. Respondents who completed an initial anonymous cross-sectional questionnaire in April were sent two further questionnaires during May and June 2020. A total of 821, 619 and 456 women pregnant following ART completed a first survey in April and subsequent surveys in May and June, respectively. A further 2684, 1965 and 1463 women seeking infertility treatment completed the surveys in the same sequence. During the first surge of the pandemic in New England, United States, 30% of pregnant and 34% of non-pregnant women reported a pre-existing diagnosis of anxiety (p=0.02) with 6% of pregnant and 11% of non-pregnant women taking anxiolytic medication (p<0.001). Pregnant respondents reported higher levels of anxiety (p=0.02) but lower levels of sadness (p=0.001) than non-pregnant patients, and more non-pregnant women were taking antidepressant medication (p=0.001). More pregnant patients cited COVID-19 as their most significant stressor in surveys 1 (52%) and 2 (39%) than non-pregnant patients still seeking infertility treatment in surveys 1 (37%) and 2 (25%; both p<0.001). Further, at survey 2, pregnant patients reported feeling significantly less sad (p<0.001) but the anxiety levels did not differ between groups (p=0.5). On survey 3, pregnant patients reported significantly less anxiety (p=0.02) and sadness (p<0.001); levels of loneliness did not differ. Pregnant patients reported that their job was their top stressor while infertility patients rated infertility as the most stressful aspect of their lives (p<0.001). Pregnant participants were more concerned about becoming infected with Covid-19 during pregnancy than non-pregnant participants (p<0.001) and about a poor pregnancy outcome due to Covid-19 infection (p<0.001). COVID-19 had a significant psychological impact on women pregnant following ART and, over the first few months of the pandemic, it remained their top stressor. However, women still experiencing infertility ranked infertility as a greater stressor than the pandemic. This may have been because infertility patients were unable to receive treatment during the study period.

To evaluate the efficiency of sperm selection by PICSI on males with different levels of abnormal sperm DNA fragmentation (SDF). A retrospective cohort study included 630 couples undergoing ICSI from October 2016 to October 2019. Cases were divided according to their SDF levels into: (Mild: 20.4-25%, Moderate: 25.1-30%, and Severe: >30%) and normal SDF (<20% control) groups. The study included females ≤37 years old, with a minimum of 5 mature oocytes injected and had a fresh blastocyst transfer. All males SDF levels were assessed prior to their ICSI cycles using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. Comparisons included pre-implantation embryo development, pregnancy, and live birth rates. The Chi square test was used for the qualitative parameters and Kruskal Walis test for non-parametric quantitative parameters (SPSS version 23). We found no significant differences between the control and high SDF groups in: type of infertility, female age, male age, count, motility, abnormal morphology, smoking status, abstinence days, No. of retrieved oocytes, and No. of mature oocytes. The outcomes of ICSI versus PICSI for the different study groups are shown in Table 1.Tabled 1Study outcomesNormal SDF n=291PICSIp-valuesMild n=147Moderate n=93Severe n=99Cleavage rate (%)75.6±18.275.3±16.677±17.978.8±14.80.6Blastocyst rate (%)64.7±19.663.2±2067.1±17.764.8±18.10.82Good quality Blastocyst rate (%)37.2±23.838±27.233±22.833.8±25.10.27Fair quality Blastocyst rate (%)17±17.115.2±18.215.2±15.618.4±17.30.5Vitrified Blastocysts3.36±3.53.37±3.52.64±2.22.67±2.70.65Implantation rate (%)44.5±43.241.5±40.738.4±44.434.4±39.20.09Clinical pregnancy rate (%)*64.461.355.760.20.53Ongoing pregnancy rate (%)*54.645.245.252.30.22Miscarriage rate (%)*14.523.515.68.20.11Live birth rate (%)*53.64343.951.20.16Data are mean (SD), otherwise stated. n refers to the number of cycles. PICSI = physiological intracytoplasmic sperm injection. SDF = Sperm DNA fragmentation. * Data are percentages only. P values <0.05 are considered significant. Open table in a new tab PICSI can perform with the same efficiency at all levels of abnormal SDF, improving their reproductive outcomes to the level of the control group. A prospective randomized trial with a control of abnormal SDF is planned as a future study to confirm these findings.

Factors present in the semen such as Zinc (Zn), white blood cells (WBC), Round (RC) and epithelial cells (EC) may increase the levels of oxidative stress (OS) and consequently, the formation of oxidative stress adducts (OSA) which may interact with sperm key biomolecules such as complement regulatory proteins required (CRP) for acrosome reaction and DNA producing sperm defective function. The aims of this study were (i) to determine if the levels of OSA are associated with the concentrations of Zn, WBC, RC, and EC and (ii) to correlate if those OSA levels induce both the increased of DNA fragmentation and loss of expression of (CRP). Retrospective. Frozen semen samples from 186 men were evaluated for sperm, WBC, RC and EC concentrations by microscopy. Oxidative Stress (OSA) and Zinc (μM) were determined by spectrophotometry. The DFI (%), HDS (%), WBC (CD45) X 10⁶/ml and Expression of CD46 (CRP) were measured by flow cytometry. For the data analysis, the samples were classified into 3 OSA categories: Normal 3.8 ≥ (n=31), BL 3.81-4.4 (n=53) and Abnormal >4.4 (n=102). The statistical analysis was performed by ANOVA sigma stat (p<0.01). The sperm DFI scores were increased both BL and Abnormal categories when were compared to Normal OSA (Normal 12.75 ± 3.39 BL 24.45 ± 2.19 Abnormal 38.59 ± 8.16 p<0.001). The concentrations of leukocytes were different for BL 0.8 ± 0.2 and Abnormal 6.8 ± 2.3 when were compared to Normal 0.4 ± 0.1 (p<0.01). The values of CRP measured as expression of CD46 indicated that BL 12.73 ± 3.07 and Abnormal 11.32 ± 4.07 are significant different when compared to Normal 15.48 ± 5.18 (p<0.05). Although no differences were found an increasing tendency are observed when the OSA levels are increasing for Zn (Normal 241.1 ± 17 μM, BL 256.4 ±19 μM and Abnormal 261.4 ± 18.4 μM), RC (Normal 3.06 ± 1.55, BL 4.55 ±3.67 and Abnormal 5.26 ± 5.74 x 10⁶ /ml), HDS (Normal 5.06 ± 2.12, BL 8.72 ± 3.27 and Abnormal 9.45 ± 3.60) and no differences were found among the 3 OSA groups for sperm and EC concentrations. The levels of oxidative stress measured as (OSA) are associated with DNA fragmentation (DFI) and the loss of the expression of complement protein (CD46). These results also suggest a possible role for leukocytes in generating oxidative stress, and demonstrate the application of CD45 staining and flow cytometry to identify leukocytes.

Despite its shortcomings, semen analysis is a cornerstone of the male infertility evaluation. As fertility care has become more accessible and patient volume has increased exponentially, it is unclear how semen parameters have changed over time, if at all. A recent meta-analysis suggests that sperm concentration has declined (Levine et al 2017). Trends in semen analysis may reflect anthropomorphic changes in patients over time, overall health status, improved access to care, or other factors. We sought to examine the trend in semen parameters over time from 1993 to 2017 at a single IVF center with mandated insurance coverage in the United States. Semen analyses collected at a single IVF clinic from September 1993 through November 2017 were evaluated, with a total of 44,048 samples analyzed. Semen analysis methodology and personnel remained constant. Specifically, semen concentration, total motility, and morphology were examined. Semen concentration was further analyzed by dividing into the following sub-groups: azoospermia, severe oligospermia (concentration 0.1-5 mil/mL), oligospermia (5.1-20 mil/mL), and normospermia (>20 mil/mL). Data were pooled and means calculated and compared across three-month long periods. SPSS was used to create one-way ANOVAs to compare means and regression analyses to evaluate changes over time. Contrary to recent studies showing a significant decline in sperm concentration, we found no significant change in concentration over time in all samples. However, when evaluating mean semen concentration of the sub-groups, we found that while the number of normospermic samples remained stable over time, the number of cases of azoospermia increased (correlation coefficient, R=0.61). Severe and moderate oligospermia remained relatively stable, with a slight decrease in case number over the study period (R=0.21, R=0.42 respectively). When evaluating the overall motility of cases with >5 million/mL concentration, there was a decline in total motility. Morphology was evaluated by examining means of normal forms, which showed no significant trend; of note, starting in 1999, strict morphology with a threshold of 4% normal forms was instituted. Trends in semen analyses may shift for a variety of reasons, including age, BMI, prevalence of male factor infertility, and lifestyle factors. Our data, however, show that semen parameters have remained relatively stable over nearly 30 years. The results of our equally large study are in direct contrast to those found by Levine et al. Given that our center is in a state that provides insurance coverage for fertility care, it may be that our results reflect a more diverse population of patients, thereby reducing selection bias. Regardless, it is important to recognize that any results from an infertility population may not reflect the male population as a whole.

The fetal origins hypothesis suggests some diseases originate in utero owing to fetal environmental adaptations. This allows live birth weights (LBW) to be a surrogate marker of in utero environmental encounters. It has been shown that babies born from IVF in a thaw cycle have higher average LBW than those from a fresh cycle and hypothesized that embryo culture media impacts LBW. Clinical IVF practices including stimulation protocols, medications, and transfer have evolved since IVF's inception. The IVF laboratory, incubators, culture media, culture devices and embryo micromanipulation have also changed over time. We demonstrated that LBW from fresh transfers is 180g lower than that from a frozen transfer, but has remained constant over an 18-year period. To investigate the association between singleton LBW in a large IVF practice over time in an 18-year period after autologous in vitro fertilization (IVF) in fresh and frozen cycles. Retrospective cohort study of 7332 singleton live births from patients who underwent autologous fresh (n=6265) or frozen (n=1030) IVF cycles at Boston IVF between 1996 and 2013. One-way ANOVA and t-tests compared average LBW in fresh and frozen cycles, LBW per cycle type over time, and ICSI versus standard IVF. LBW was divided into extremely low, very low, and low birth weight categories and fresh versus frozen transfer LBW were compared via chi-square test. Linear regression was performed to assess for LBW correlation with clinical characteristics. ANOVA was performed across known changes. Chi-square revealed neonates from fresh cycles have a 9.3% risk of low birth weight versus 5.6% from frozen cycles (p<0.001). The number of low birth weight babies did not change over time. No difference in LBW was noted between ICSI and standard IVF, p=0.954. Linear regression revealed fresh transfer LBW is correlated directly with maternal parity and inversely with peak Estradiol. Male neonates weighed an average of 122g more than females in fresh and frozen cycles, p<0.05. No difference was found across changes of media, laboratory location, or gonadotropins. Changes in laboratory procedures and clinical care did not impact LBW over the study period. The impact of fresh versus frozen embryo transfer outweighs any aff5ct clinical or laboratory changes may have on LBW.

Mitochondrial function is essential for reproduction. We hypothesized that the decreased viability of aging oocytes may be partly due to impaired mitochondrial function. We investigated key mitochondrial stress parameters in association with aging in mouse oocytes, and assessed mtDNA quantity and NADH/FAD fluorescence as potential invasive and non-invasive biomarkers of age-related changes, respectively. Experimental study. Mature (metaphase II) ocytes from old (12 months) and young (9 weeks) C57BL/6J mice were compared. Metabolic stress was assessed by determining the levels of reactive oxygen species (ROS) under baseline conditions and following H2O2 treatment (using carboxy-H2DCFDA fluorescent staining); and by quantifying expression of mitochondrial unfolded protein response (mt-UPR) genes (Clpp, Dnaja3, Haspd1, Haspe1) via qRT-PCR. Absolute mtDNA levels were quantified via cloning of mitochondria specific gene (Cox3) as a standard, followed by qPCR analysis of individual oocytes (20 young and 20 old). Fluorescence lifetime imaging microscopy (FLIM) was used to obtain non-invasive measurements of intracellular NADH and FAD. FLIM measurements were performed on individual oocytes (19 young and 14 old) in an on-stage incubator system to avoid environmental effects. Oocytes assessed by FLIM also underwent mtDNA quantification as described above. ROS levels in aged MII oocytes were higher following pretreatment with H2O2 (p<0.05). The expression of mt-UPR gene Hspd1 was also elevated in aged MII oocytes (p<0.05). Oocytes from old mice had significantly less mtDNA compared to young ones (58,222 +/- 15,429 copies/oocyte vs. 162,106 +/- 19,302 copies/oocyte [mean +/- SEM; p<0.01]). FLIM analysis showed a weak but statistically significant correlation with age and mtDNA copy number (p<0.05). Aging is associated with a significant increase in ROS levels in oocytes under stressful conditions and elevated expression of mitochondrial stress response gene Hspd1. Importantly, aged mouse oocytes have lower mtDNA levels that correlate with NADH/FAD levels detected by FLIM. Further delineation of mitochondrial changes associated with ageing may help the development of diagnostic biomarkers and therapeutic tools in assisted reproduction.

Purpose The aim of this study is to evaluate the outcomes of in vitro fertilization (IVF), including cumulative live birth rate, among women <25 years, 25 to <30 years, and 30 to <35 years. Methods A retrospective cohort study of all women 18 to <35 years of age at their first fresh-embryo, non-donor IVF cycle from January 1995 through December 2012 at a single center was conducted. A competing-risk regression model was used to estimate the cumulative probability and 95 % confidence interval (CI) of the first live birth in up to 6 cycles during the study period with IVF cycle number as the time metric. Results Among 7243 women who underwent 16,792 cycles, there were 163 (2.3 %) women <25 years, 1691 (23.3 %) women 25 to <30 years, and 5389 (74.4 %) women 30 to <35 years. Women <25 years had the lowest cumulative live birth rate after each cycle, followed by women 30 to <35 years. In both groups, the cumulative live birth rate after 6 cycles was significantly lower than that of women 25 to <30 years; these rates were 58 % (95 % CI 0.51–0.66) among women <25 years, 69 % (95 % CI 0.67–0.71) among women 25 to <30 years, and 64 % (95 % CI 0.63–0.65) among women 30 to <35 years. Conclusions Our findings are consistent with other reports of less favorable IVF treatment outcomes in women <25 years of age following their first IVF cycle. This indicates that there are underlying factors in couples with a female <25 years of age that should lead to different treatment counseling when they attempt IVF.