STUDY QUESTION:Are relative mitochondrial DNA (mtDNA) content and mitochondrial genome (mtGenome) variants in human cumulus cells (CCs) associated with oocyte reproductive potential and assisted reproductive technology (ART) outcomes?SUMMARY ANSWER:Neither the CC mtDNA quantity nor the presence of specific mtDNA genetic variants was associated with ART outcomes, although associations with patient body mass index (BMI) were detected, and the total number of oocytes retrieved differed between major mitochondrial haplogroups.WHAT IS KNOWN ALREADY:CCs fulfil a vital role in the support of oocyte developmental competence. As with other cell types, appropriate cellular function is likely to rely upon adequate energy production, which in turn depends on the quantity and genetic competence of the mitochondria. mtDNA mutations can be inherited or they can accumulate in somatic cells over time, potentially contributing to aging. Such mutations may be homoplasmic (affecting all mtDNA in a cell) or they may display varying levels of heteroplasmy (affecting a proportion of the mtDNA). Currently, little is known concerning variation in CC mitochondrial genetics and how this might influence the reproductive potential of the associated oocyte.STUDY DESIGN, SIZE, DURATION:This was a prospective observational study involving human CCs collected with 541 oocytes from 177 IVF patients. mtDNA quantity was measured in all the samples with a validated quantitative PCR method and the entire mtGenome was sequenced in a subset of 138 samples using a high-depth massively parallel sequencing approach. Associations between relative mtDNA quantity and mtGenome variants in CCs and patient age, BMI (kg/m2), infertility diagnosis and ART outcomes were investigated.PARTICIPANTS/MATERIALS, SETTING, METHODS:Massively parallel sequencing permitted not only the accurate detection of mutations but also the precise quantification of levels of mutations in cases of heteroplasmy. Sequence variants in the mtDNA were evaluated using Mitomaster and HmtVar to predict their potential impact.MAIN RESULTS AND THE ROLE OF CHANCE:The relative mtDNA CC content was significantly associated with BMI. No significant associations were observed between CC mtDNA quantity and patient age, female infertility diagnosis or any ART outcome variable. mtGenome sequencing revealed 4181 genetic variants with respect to a reference genome. The COXI locus contained the least number of coding sequence variants, whereas ATPase8 had the most. The number of variants predicted to affect the ATP production differed significantly between mitochondrial macrohaplogroups. The total number of retrieved oocytes was different between the H-V and J-T as well as the U-K and J-T macrohaplogroups. There was a non-significant increase in mtDNA levels in CCs with heteroplasmic mitochondrial mutations.LARGE SCALE DATA:N/A.LIMITATIONS, REASONS FOR CAUTION:Although a large number of samples were analysed in this study, it was not possible to analyse all the CCs from every patient. Also, the results obtained with respect to specific clinical outcomes and macrohaplogroups should be interpreted with caution due to the smaller sample sizes when subdividing the dataset.WIDER IMPLICATIONS OF THE FINDINGS:These findings suggest that the analysis of mtDNA in CCs is unlikely to provide an advantage in terms of improved embryo selection during assisted reproduction cycles. Nonetheless, our data raise interesting biological questions, particularly regarding the interplay of metabolism and BMI and the association of mtDNA haplogroup with oocyte yield in ovarian stimulation cycles.STUDY FUNDING/COMPETING INTEREST(S):This study was funded by National Institutes of Health grant 5R01HD092550-02. D.J.N. and C.R. co-hold patent US20150346100A1 and D.J.N. holds US20170039415A1, both for metabolic imaging methods. D.W. receives support from the NIHR Oxford Biomedical Research Centre. The remaining authors have no conflicts of interest to declare.

OBJECTIVE:To determine whether fluorescence lifetime imaging microscopy (FLIM) detects differences in metabolic state among cumulus cell samples and whether their metabolic state is associated with patient age, body mass index (BMI), and antimüllerian hormone (AMH) level and maturity of the oocyte. DESIGN:Prospective observational study. SETTING:Academic laboratory. PATIENT(S):Cumulus cell (CC) clusters from cumulus-oocyte complexes were collected from patients undergoing assisted reproductive technology treatment after oocyte retrieval and vitrified. INTERVENTION(S):Cumulus cell metabolism was assessed using FLIM to measure autofluorescence of nicotinamide adenine (phosphate) dinucleotide and flavine adenine dinucleotide, endogenous coenzymes essential for cellular respiration and glycolysis. Patient age, BMI, and AMH level and the maturity of the corresponding oocytes were recorded. MAIN OUTCOME MEASURE(S):Quantitative information from FLIM was obtained regarding metabolite concentrations from fluorescence intensity and metabolite enzyme engagement from fluorescence lifetimes. Associations were investigated between each FLIM parameter and oocyte maturity and patient age, BMI, and AMH. Variance between CC clusters within and between patients was determined. RESULT(S):Of 619 CC clusters from 193 patients, 90 were associated with immature oocytes and 505 with metaphase II oocytes. FLIM enabled quantitative measurements of the metabolic state of CC clusters. These parameters were significantly correlated with patient age and AMH independently, but not with BMI. Cumulus cell nicotinamide adenine (phosphate) dinucleotide FLIM parameters and redox ratio were significantly associated with maturity of the enclosed oocyte. CONCLUSION(S):FLIM detects variations in the metabolic state of CCs, showing a greater variance among clusters from each patient than between patients. Fluorescence lifetime imaging microscopy can detect CC metabolic associations with patient age and AMH and variations between mature and immature oocytes, suggesting the potential utility of this technique to help identify superior oocytes.

To investigate the association between Fluorescence Lifetime Imaging Microscopy (FLIM) of metabolic function in cumulus cells and 1) oocyte maturity; and 2) fertilization potential. The over-arching objective of this work is to determine whether metabolic function of cumulus cells can non-invasively detect maturity and fertilization of the corresponding oocytes. And to determine whether these metabolic parameters are correlated with maternal age and BMI. Prospective observational study. Cumulus cell clusters (n=422 from 212 patients; mean age 35.9 y, mean BMI 26.9 kg/m2) were dissected from the cumulus oocyte complexes prior to assisted reproduction treatments. Maturity and fertilization status of the corresponding enclosed oocytes were tracked. 60 samples were associated with immature oocytes and 362 with mature oocytes. 282 oocytes resulted in 2 pronuclei zygotes and 36 oocytes did not fertilize. Cumulus cell metabolic function was assessed non-invasively using FLIM to measure the autofluorescence of NADH and FAD+, endogenous coenzymes essential for cellular respiration and glycolysis. Quantitative information was obtained on 1) metabolite concentrations from fluorescence intensity; and 2) metabolite enzyme engagement from fluorescence lifetimes. Each FLIM measurement provides a total of 9 quantitative metabolic parameters (4 for NADH, 4 for FAD+ and Redox Ratio: NADH intensity /FAD+ intensity). These cumulus cell metabolic parameters were compared with oocyte maturity, oocyte fertilization status and patient age and BMI using multi-level generalized linear models providing information of cumulus variance within and between patients. We found statistically significant correlations regarding both cumulus cell NADH metabolic parameters (p<0.05) and the Redox Ratio (p=0.001) and oocyte maturity. FAD+ intensity and fraction bound to enzyme displayed significant variation between cumulus cells associated with fertilized compared to unfertilized oocytes (p<0.05). All metabolic parameters of cumulus cells were significantly correlated with maternal age (p<0.05), but not with BMI. Metabolic imaging can non-invasively detect significant metabolic variations in cumulus cells between mature and immature oocytes, suggesting the potential utility of this technique in a clinical setting. These metabolic parameters were also associated with oocyte fertilization potential and clinically relevant factors, such as maternal age, but not BMI. Moreover, we are in the process of developing machine learning based algorithms combining patient characteristics and cumulus cell FLIM data to predict the maturity of the corresponding oocyte.

Bidirectional metabolic cooperativity between the human oocyte and its surrounding cumulus cells is essential for successful development. However, the relationship between cumulus cell metabolism and oocyte viability is not well established. Our aim was to determine whether non-invasive metabolic imaging of cumulus cells mitochondrial function is associated with the clinical outcome of the corresponding oocyte. Prospective observational study. Cumulus cell clusters (n=458 from 177 patients, mean age 36.7y) were dissected from cumulus oocyte complexes prior to assisted reproduction technique treatment. Morphology of embryos was assessed on day 3. Grades were stratified as excellent (n=89), good (n=79), fair (n=43) and poor morphology (n=62). Clinical outcomes of the corresponding oocytes were tracked. Embryos were transferred on day 3 or 5, either fresh (n=48 and 10) or after warming (n=7 and 20). Cumulus cell metabolism was assessed non-invasively using fluorescence lifetime imaging microscopy (FLIM) to measure the autofluorescence of NADH and FAD+, two key metabolites in cellular respiration and glycolysis. This approach enabled quantitative information to be obtained on concentrations of these coenzymes and on metabolite enzyme engagement. Overall a single FLIM measurement provides a total of 8 metabolic parameters (4 for NADH and 4 for FAD+). An additional parameter, the Redox Ratio can also be acquired (NADH intensity / FAD+ intensity). We used multilevel models to investigate the association of cumulus cell metabolic parameters with day 3 embryo morphology and clinical outcome. Of the cumulus samples analyzed, 62 corresponded to embryos that did not implant, and 23 led to a clinical pregnancy. We found significant associations between cumulus cell FAD+ fraction bound to enzyme (p =0.02) and FAD+ short lifetime (p <0.001) and the clinical outcome of the corresponding embryo. There were no significant associations between cumulus metabolic parameters and embryo quality assessed at day 3. Cumulus cell metabolic parameters are significantly associated with embryo outcome, although not with day 3 embryo quality. These findings suggest that metabolism and morphology provide complementary information. We are working to develop an embryo selection algorithm using machine learning based approaches that combines cumulus cell metabolic data, patient clinical characteristics (age, BMI, AMH levels), and morphological assessment of embryo quality.

Cumulus cells (CCs) fulfil a vital role in support of oocyte developmental competency. The appropriate function of CCs relies on adequate energy production, which in turn depends on the quantity and genetic competence of mitochondria. We sought to characterize the mitochondrial DNA content of a large number of human CCs samples via a combination of quantitative (relative mtDNA copy number) and qualitative (DNA sequence) analyses. Results were considered in relation to factors of relevance to oocyte reproductive potential, such as female age and body mass index (BMI), as well as to embryo characteristics and assisted reproductive technology (ART) outcomes. Prospective observational study. Human CCs, associated with 347 oocytes from 110 IVF patients, were recruited in the ongoing study. Patient age, body mass index (BMI) (kg/m2), clinical diagnosis and ART outcomes were recorded with respect to each cumulus complex. Mitochondrial DNA quantity was measured in all the samples with a validated quantitative PCR method and the entire mitochondrial genome was sequenced in a subset of 119 samples using a high-depth massively parallel sequencing approach. Sequence variants in the mtDNA were evaluated using Mitomaster and HmtVar to predict their potential impact. Generalized linear mixed model and other appropriate statistical models were used to assess significance (P≤0.05). Mitochondrial genome sequencing of CCs samples showed a total of 952 synonymous (i.e. no amino acid change) single nucleotide variants (SNVs) and another 626 non-synonymous (altered amino acid sequence) SNVs. Most SNVs were homoplasmic and therefore presumed to have been inherited, but 21 were heteroplasmic variants and potentially had a somatic origin. 104 of the homoplasmic variants, detected in 63 samples, were predicted to impact protein function, potentially affecting ATP production. Significant associations were observed between BMI and the number of non-synonymous mtDNA variants (p=0.037) and BMI and mtDNA quantity (P = 0.013). Moreover, a significant difference was also detected between mtDNA levels in CCs and normal weight (BMI 18.5-24.9) and obesity-class groups (BMI-30.0-34.9, P=0.023; ≥40.0, P=0.024). No significant differences were observed between mtDNA quantity in CCs and oocyte fertilization status or embryo quality. No significant associations were detected between cumulus cell mtDNA quantity or sequence variants and clinically relevant endpoints. These findings suggest that, analysis of mtDNA (sequence and quantity) in CCs is unlikely to provide an advantage in terms of improved embryo selection during assisted reproduction cycles. Nonetheless, our data raise interesting biological questions, particularly regarding the interplay of metabolism and BMI.

Male subfertility is often associated with sub-optimal health status and traditional semen and hormone analysis reveal only limited information about the reduced fertility potential. Circulating small non-coding RNAs (sncRNAs) are paracrine and endocrine messengers, with prognostic potential. Here, we utilised small RNA-Seq to identify novel cell-free circulating sncRNAs that could act as potential biomarkers of male subfertility. We analysed sera from twelve subfertile men and four controls. The subfertile men were further sub-divided into the three groups based on reproductive hormone levels: group 1 (n = 4): hormone levels similar to the controls, group 2 (n = 4) showing elevated FSH levels, and group 3 (n = 4) with low total testosterone (TT). Total RNA was extracted from serum and sequenced to identify miRNAs and piRNAs. Selected sncRNAs were qPCR validated in a larger and independent cohort of subfertile men (n = 57) and normozoospermic controls (n = 19). RNA-Seq resulted in the identification of 1123 and 330 circulating miRNAs and piRNAs, respectively. Several miRNAs and piRNAs were differentially (p = 0.05) present between controls and subfertile men. Subfertile men with low TT appeared to have a distinct sncRNA profile, compared to group 1 and 2. Validation of two miRNAs (hsa-miR-542-5p and hsa-let-7i-3p) and one piRNA (hsa-piR-26399) in an independent cohort confirmed a significant difference in circulating levels between subfertile and control men. Enrichment analysis of the putative miRNA targets showed association with steroid biosynthesis pathway highlighting a potential regulatory role of these miRNAs. We propose that circulating sncRNAs may represent new important functional biomarkers in male reproductive endocrinology.

Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n = 19 for alkaline and 13 for neutral group) and lymphoma (n = 13 for alkaline and 09 for neutral group) compared with fertile donors (n = 20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p < 0.001) and neutral (7.5% vs. 13.4%; p < 0.05) Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p < 0.05) Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment.

Cryoinjury is a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, morphology, and viability. This study was designed to identify potential proteomic changes in human sperm cells throughout the cryopreservation process. Comparisons made within this study included the detection of the sperm proteomic changes induced by incubation of the sperm cells with a protein-free cryoprotectant (with and without CryoSperm), and the proteomic changes induced by freezing, thawing, and subsequent after-thawing incubation at two different temperatures (0 °C vs. 23 °C). Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for protein quantification. LC-MS/MS resulted in the identification of 769 quantifiable proteins. The abundance of 105 proteins was altered upon CryoSperm incubation. Freezing and thawing also induced substantial protein changes. However, fewer changes were observed when semen was thawed and then maintained after-thawing at approximately 0 °C than when it was maintained after-thawing at 23 °C, with 60 and 99 differential proteins detected, respectively, as compared to unfrozen semen incubated in CryoSperm. Collectively, these differences indicate that substantial changes occur in the sperm proteome at every stage of the cryopreservation process which may ultimately impair the sperm fertilizing capability. This is the first study to compare protein levels in fresh and cryopreserved semen using the TMT technology coupled to LC-MS/MS.

As a number of children born by assisted reproductive technology (ART) are increasing each year across the developed world, the health of such offspring is a matter of public concern. Does the integrity of the paternal genome impact on offspring health In societal terms, as birth rates fall, and the Western population become unsustainable, do the benefits outweigh the costs of creating and providing for this ART conceived subpopulation There are little data to date to answer these questions. The long-term health of such children has largely been ignored, and success measured only by early (prebirth) outcomes such as embryo quality or pregnancy. However, there are powerful paradigms such as ageing and smoking that give vital clues as to the potential impact of unhealthy spermatozoa on disease risk, mental and physical health, fertility and mortality of these offspring.

The integrity of the sperm genome and epigenome are critical for normal embryonic development. The advent of assisted reproductive technology has led to an increased understanding of the role of sperm in fertilization and embryogenesis. During fertilization, the sperm transmits not only nuclear DNA to the oocyte but also activation factor, centrosomes, and a host of messenger RNA and microRNAs. This complex complement of microRNAs and other non-coding RNAs is believed to modify important post-fertilization events. Thus, the health of the sperm genome and epigenome is critical for improving assisted conception rates and the birth of healthy offspring.

Standard semen parameters are poor predictors of fertility potential. To date, apart from, paternal karyotyping sperm factors are not evaluated in recurrent pregnancy loss (RPL), only recent studies have emphasized the role of sperm factors in early embryonic development as sperm transcribes genes critical for early embryonic development. Sperm DNA integrity is useful diagnostic and prognostic marker and has clinical implications in idiopathic recurrent pregnancy loss (iRPL) following spontaneous conception. The aim of this study was to assess DNA integrity in cases experiencing iRPL following spontaneous conception.Semen samples from 45 patients and 20 controls were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was used to measure DNA fragmentation index (DFI).By applying receiver operating curve (ROC) analysis, sperm DFI of approximately 26 % was found in male partner of couples experiencing iRPL.Our data indicate that sperm from men with a history of iRPL have a higher percentage of DNA damage as compared to control group, and this can explain pregnancy loss in these patients. Men with higher DFI are infertile whereas men with lower DFI (26 %) are able to conceive but experience recurrent pregnancy loss. Thus it is important to evaluate sperm DFI in couples experiencing iRPL to understand exact aetiology of RPL and determine prognosis and management.

ObjectiveRecurrent pregnancy loss affects approximately 3-5% percent of couples attempting to have a child. Despite extensive evaluation majority of couple experiencing recurrent pregnancy loss (RPL), have no identifiable factors (iRPL). The aim of this study was to analyze the sperm transcripts level in cases of iRPL.DesignThe present study was set up to examine the level of relationship between sperm transcripts (PRM2, WNT5A, PSG1, ZNF582, HSP90) and iRPL. Fifty couples who experienced RPL and 30 fertile controls were enrolled in the study. Semen samples were collected by masturbation from after 4 days of abstinence. Semen analysis was performed as per WHO (1999) guidelines.Materials and MethodsTotal RNA was isolated from 1×107 spermatozoa using RNeasy mini kit. RNA was reverse-transcribed into complementary DNA using random hexamer priming and Revert AidTM M-MuLv reverse transcriptase. Quantitative analysis of each gene was performed by CFX96 real Time System (Bio-Rad, USA) using SsoFastTM EvaGreen Supermix (Bio-Rad Laboratories, CA). The relative quantification of target genes normalized to β-actin was calculated by 2ΔCt method. The relative gene expression levels between control and patient subjects were compared by student- t test. If the P value was ≤0.05, the difference was considered to be statistically significant.ResultsiRPL and control groups did not show any significant differences in the sperm parameters. There was a significant difference between fertile and iRPL group for PSG1, PRM2 ZNF582 and HSP90. However, no significant difference was found for WNT5A between fertile and iRPL patients.ConclusionThese findings, suggests that sperm PSG1, PRM2 ZNF582 and HSP90 mRNA (but not WNT5A) in the egg after fertilization, lead to the conclusion that these transcripts may have a functional role during early embryo development and/or implantation. However, its functional implication may require further validation. ObjectiveRecurrent pregnancy loss affects approximately 3-5% percent of couples attempting to have a child. Despite extensive evaluation majority of couple experiencing recurrent pregnancy loss (RPL), have no identifiable factors (iRPL). The aim of this study was to analyze the sperm transcripts level in cases of iRPL. Recurrent pregnancy loss affects approximately 3-5% percent of couples attempting to have a child. Despite extensive evaluation majority of couple experiencing recurrent pregnancy loss (RPL), have no identifiable factors (iRPL). The aim of this study was to analyze the sperm transcripts level in cases of iRPL. DesignThe present study was set up to examine the level of relationship between sperm transcripts (PRM2, WNT5A, PSG1, ZNF582, HSP90) and iRPL. Fifty couples who experienced RPL and 30 fertile controls were enrolled in the study. Semen samples were collected by masturbation from after 4 days of abstinence. Semen analysis was performed as per WHO (1999) guidelines. The present study was set up to examine the level of relationship between sperm transcripts (PRM2, WNT5A, PSG1, ZNF582, HSP90) and iRPL. Fifty couples who experienced RPL and 30 fertile controls were enrolled in the study. Semen samples were collected by masturbation from after 4 days of abstinence. Semen analysis was performed as per WHO (1999) guidelines. Materials and MethodsTotal RNA was isolated from 1×107 spermatozoa using RNeasy mini kit. RNA was reverse-transcribed into complementary DNA using random hexamer priming and Revert AidTM M-MuLv reverse transcriptase. Quantitative analysis of each gene was performed by CFX96 real Time System (Bio-Rad, USA) using SsoFastTM EvaGreen Supermix (Bio-Rad Laboratories, CA). The relative quantification of target genes normalized to β-actin was calculated by 2ΔCt method. The relative gene expression levels between control and patient subjects were compared by student- t test. If the P value was ≤0.05, the difference was considered to be statistically significant. Total RNA was isolated from 1×107 spermatozoa using RNeasy mini kit. RNA was reverse-transcribed into complementary DNA using random hexamer priming and Revert AidTM M-MuLv reverse transcriptase. Quantitative analysis of each gene was performed by CFX96 real Time System (Bio-Rad, USA) using SsoFastTM EvaGreen Supermix (Bio-Rad Laboratories, CA). The relative quantification of target genes normalized to β-actin was calculated by 2ΔCt method. The relative gene expression levels between control and patient subjects were compared by student- t test. If the P value was ≤0.05, the difference was considered to be statistically significant. ResultsiRPL and control groups did not show any significant differences in the sperm parameters. There was a significant difference between fertile and iRPL group for PSG1, PRM2 ZNF582 and HSP90. However, no significant difference was found for WNT5A between fertile and iRPL patients. iRPL and control groups did not show any significant differences in the sperm parameters. There was a significant difference between fertile and iRPL group for PSG1, PRM2 ZNF582 and HSP90. However, no significant difference was found for WNT5A between fertile and iRPL patients. ConclusionThese findings, suggests that sperm PSG1, PRM2 ZNF582 and HSP90 mRNA (but not WNT5A) in the egg after fertilization, lead to the conclusion that these transcripts may have a functional role during early embryo development and/or implantation. However, its functional implication may require further validation. These findings, suggests that sperm PSG1, PRM2 ZNF582 and HSP90 mRNA (but not WNT5A) in the egg after fertilization, lead to the conclusion that these transcripts may have a functional role during early embryo development and/or implantation. However, its functional implication may require further validation.