Early prepubertal cyclophosphamide exposure in mice results in long-term loss of ovarian reserve, and impaired embryonic development and blastocyst quality.

BackgroundDue to improved treatment, there is an increasing focus on the reproductive potential of survivors of childhood cancer. Cytotoxic chemotherapy accelerates the decline in the number of primordial follicles within the mammalian ovary at all ages, but effects on the developmental potential of remaining oocytes following prepubertal cancer treatment are unclear.ObjectivesTo investigate whether cyclophosphamide (CY) exposure in the prepubertal period in female mice influences ovarian function and the functional competence of oocytes in adulthood.MethodsThis study used Swiss albino mice as the experimental model. Female mice were treated with 200 mg/kg CY on either postnatal day 14 (CY14), 21 (CY21) or 28 (CY28) i.e at a prepubertal and 2 young postpubertal ages. At 14 weeks of life, ovarian function, functional competence of oocytes, and embryo quality were assessed.ResultsThe number of primordial follicles decreased significantly in CY14 and CY21 groups compared to control (p ConclusionOur results indicate long-term effects on the developmental competence of oocytes exposed to CY in early but not adult life. These data provide a mechanism whereby long-term fertility can be impaired after chemotherapy exposure, despite the continuing presence of follicles within the ovary, and support the need for fertility preservation in prepubertal girls before alkylating agent exposure

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment

Spent embryo culture medium metabolites are related to the in vitro attachment ability of blastocysts

The metabolomic profile of an embryo culture medium can aid in the advanced prediction of embryonic developmental potential and genetic integrity. But it is not known if this technology can be used to determine the in vitro potential of inner cell mass (lCM) in adherence and proliferation. Here, we investigated the developmental potential of mouse 2-cell embryos carrying cisplatin-induced DNA lesions (lDL), beyond blastocyst stage using ICM outgrowth assay. The genetic integrity of ICM cells was determined by comet assay. The metabolic signatures of spent medium were recorded 84 hours post injection of hCG (hpi- hCG), and after 96 hours of extended in vitro culture (Ex 96) by NMR spectroscopy. We observed that blastocysts that lack the ability to adhere in vitro had an increased requirement of pyruvate (p < 0.01), lactate (p < 0.01), and were accompanied by a significant reduction of pyruvatealanine ratio in the culture medium. We propose that the aforementioned metabolites from 84 hpi-hCG spent medium be further explored using appropriate experimental models, to prove their potential as biomarkers in the prediction of implantation ability of in vitro derived human embryos in clinical settings

Influence of sperm DNA damage on human preimplantation embryo metabolism

Understanding the embryo metabolic response to sperm induced specific abnormalities could help in developing the metabolic markers to prevent the transfer of embryos carrying sperm mediated defects. In this study, NMR based metabolic profiling of the embryo spent media was employed in 34 patients undergoing ICSI cycles. Processed ejaculates were tested for DNA damage using comet assay. Relative intensities of the metabolites from 74 embryo spent media samples from 34 patients and 23 medium controls were profiled using H-1 NMR and compared between `male-factor' and control groups. Relative intensities in the subgroups which are independent of patients with male factor or tubal factors, but related to the extent of sperm DNA damage were also compared. Sperm characteristics including DNA damage levels (Olive tail moment, OTM) were significantly different between `male-factor' and control groups (P < 0.001-0.0001). Of the metabolites analyzed, glutamine intensity was significantly lower in `male factor' group (P < 0.01) whereas, pyruvate intensity was significantly lower in embryos derived from the processed sperm fraction having <1.0 OTM (P = 0.003). In contrast glutamine and alanine intensities were significantly higher in the embryos derived from sperm population having OTM <1.0. (P = 0.03 & 0.005 respectively). Pyruvate to alanine ratio was significantly lower in <1.0 OTM group (P < 0.0001). This study indicates that increased level of sperm DNA damage in the processed ejaculate affects embryo metabolism which could be related to embryonic genetic integrity. (C) 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction 82 the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn

Semen abnormalities, sperm DNA damage and global hypermethylation in health workers occupationally exposed to ionizing radiation.

BACKGROUND: Cytogenetic studies have demonstrated that low levels of chronic radiation exposure can potentially increase the frequency of chromosomal aberrations and aneuploidy in somatic cells. Epidemiological studies have shown that health workers occupationally exposed to ionizing radiation bear an increased risk of hematological malignancies. OBJECTIVES: To find the influence of occupational radiation exposure on semen characteristics, including genetic and epigenetic integrity of spermatozoa in a chronically exposed population. METHODS: This cross sectional study included 134 male volunteers of which 83 were occupationally exposed to ionizing radiation and 51 were non-exposed control subjects. Semen characteristics, sperm DNA fragmentation, aneuploidy and incidence of global hypermethylation in the spermatozoa were determined and compared between the non-exposed and the exposed group. RESULTS: Direct comparison of the semen characteristics between the non-exposed and the exposed population revealed significant differences in motility characteristics, viability, and morphological abnormalities (P<0.05-0.0001). Although, the level of sperm DNA fragmentation was significantly higher in the exposed group as compared to the non-exposed group (P<0.05-0.0001), the incidence of sperm aneuploidy was not statistically different between the two groups. However, a significant number of hypermethylated spermatozoa were observed in the exposed group in comparison to non-exposed group (P<0.05). CONCLUSIONS: We provide the first evidence on the detrimental effects of occupational radiation exposure on functional, genetic and epigenetic integrity of sperm in health workers. However, further studies are required to confirm the potential detrimental effects of ionizing radiation in these subjects

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs

Sperm motility and DNA fragmentation analysis in relation to EA.

<p>A) Sperm motility analysis in neat ejaculate (N = 76) (■) as well as in the processed fraction (N = 32) from four study intervals. Processed fraction was incubated at 37°C and motility analysis was performed at 0h (●), 6h (▲), and 24h (▼) time interval. Please note that differences in sperm motility with corresponding EA intervals were not statistically significant. B) Box plot depicting the DNA fragmentation level as measured by the sperm chromatin dispersion (SCD) assay in the neat ejaculate (N = 56) (□) and processed fraction (N = 32) (■). ^aP < 0.05 <i>Vs</i> corresponding group in EA-5; ^bP < 0.001 <i>Vs</i> corresponding group in EA-7; ^c P < 0.01 <i>Vs</i> corresponding group in EA-7; ^dP < 0.05 <i>Vs</i> corresponding group in EA-7. C) Sperm DNA longevity analysis by SCD assay in the processed fraction at 0h (■), 1h (●), 6h (▲), and 24h (▼) time interval.</p

Basic semen characteristics in relation to different EA intervals.

<p>Basic semen characteristics in relation to different EA intervals.</p

Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity <i>In Vitro</i>

<div><p>Background</p><p>The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome.</p><p>Objective</p><p>To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status.</p><p>Methods</p><p>This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA.</p><p>Results</p><p>Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, <i>in vitro</i> incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001).</p><p>Conclusion</p><p>Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation <i>in vitro</i> should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes.</p></div

Sperm chromatin maturity and hypermethylation level at various EA periods.

<p><b>A)</b> Box plot demonstrating the percentage of aniline blue positive spermatozoa (suggestive of immature chromatin) in the neat ejaculate from four study intervals (N = 32) (^a P < 0.05 <i>Vs</i> EA-1, ^b P < 0.01 <i>Vs</i> EA-1). <b>B)</b> Box plot demonstrating the percentage of 5mC positive spermatozoa (suggestive of hypermethylation) across study intervals (N = 40). Please note that the differences were not statistically significant.</p