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

BACKGROUND: Due 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. OBJECTIVES: To investigate whether cyclophosphamide (CY) exposure in the prepubertal period in female mice influences ovarian function and the functional competence of oocytes in adulthood. METHODS: This 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. RESULTS: The number of primordial follicles decreased significantly in CY14 and CY21 groups compared to control (p < 0.01). The number of oocytes from superovulated was 8.5 ± 1.4, 24.1 ± 2.9 and 26.8 ± 2.1 in CY14, CY21 and CY28 respectively which was significantly lower than control (50.2 ± 3.2; p < 0.001). In vitro culture of CY14 embryos demonstrated only 55.4% blastocyst formation (p < 0.0001) and reduced ability of inner cell mass (ICM) to proliferate in vitro (p < 0.05) at 120 and 216 h post insemination respectively. On the other hand, ICM proliferation was unaltered in 2 young postpubertal ages. CONCLUSION: Our 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.

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 (ICM) in adherence and proliferation. Here, we investigated the developmental potential of mouse 2-cell embryos carrying cisplatin-induced DNA lesions (IDL), 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 pyruvate-alanine 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.

Epigenetic changes in preimplantation embryos subjected to laser manipulation.

The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6-8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 µs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.

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.

Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism.

Laser assisted zona hatching (LAH) is a routinely used therapeutic intervention in assisted reproductive technology for patients with poor prognosis. However, results are not conclusive in demonstrating the benefits of zona hatching in improving the pregnancy rate. Recent observations on LAH induced genetic instability in animal embryos prompted us to look into the effects of laser assisted zona hatching on the human preimplantation embryo quality and metabolic uptake using high resolution nuclear magnetic resonance (NMR) technology. This experimental prospective study included fifty embryos from twenty-five patients undergoing intra cytoplasmic sperm injection. Embryo quality assessment followed by profiling of spent media for the non-invasive evaluation of metabolites was performed using NMR spectroscopy 24 hours after laser treatment and compared with that of non-treated sibling embryos. Both cell number and embryo quality on day 3 of development did not vary significantly between the two groups at 24 hours post laser treatment interval. Time lapse monitoring of the embryos for 24 hours did not reveal blastomere fragmentation adjacent to the point of laser treatment. Similarly, principal component analysis of metabolites did not demonstrate any variation across the groups. These results suggest that laser assisted zona hatching does not affect human preimplantation embryo morphology and metabolism at least until 24 hours post laser assisted zona hatching. However, studies are required to elucidate laser induced metabolic and developmental changes at extended time periods. ABBREVIATIONS: AH: assisted hatching; ART: assisted reproductive technology; DNA: deoxy-ribo nucleic acid; LAH: laser assisted hatching; MHz: megahertz; NMR: nuclear magnetic resonance; PCA: principal component analysis; PGD: preimplantation genetic diagnosis; TLM: time lapse monitoring.

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 1H 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.

Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro.

BACKGROUND: 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. OBJECTIVE: To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. METHODS: 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. RESULTS: 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, in vitro 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). CONCLUSION: 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 in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes.

Competitive electron transfer in a novel, broad-band capturing, subphthalocyanine-AzaBODIPY-C60 supramolecular triad.

A V-configured subphthalocyanine-azaBODIPY-C60 supramolecular triad has been newly synthesized, and sequential energy and electron transfer leading to the formation of charge separated states, useful properties relevant for solar energy harvesting and building optoelectronic devices, is reported.

Multistep energy and electron transfer in a "V-configured" supramolecular BODIPY-azaBODIPY-fullerene triad: mimicry of photosynthetic antenna reaction-center events.

A new photosynthetic antenna-reaction-center model compound composed of covalently linked BF2 -chelated dipyrromethene (BODIPY), BF2 -chelated azadipyrromethene (azaBODIPY), and fullerene (C60 ), in a "V-configuration", has been newly synthesized and characterized by using a multistep synthetic procedure. Optical absorbance and steady-state fluorescence, computational, and electrochemical studies were systematically performed in nonpolar, toluene, and polar, benzonitrile, solvents to establish the molecular integrity of the triad and to construct an energy-level diagram revealing different photochemical events. The geometry obtained by B3LYP/6-31G* calculations revealed the anticipated V-configuration of the BODIPY-azaBODIPY-C60 triad. The location of the frontier orbitals in the triad tracked the site of electron transfer determined from electrochemical studies. The different photochemical events originated from (1) BODIPY* were realized from the energy-level diagram. Accordingly, (1) BODIPY* resulted in competitive ultrafast energy transfer to produce BODIPY-(1) azaBODIPY*-C60 and electron transfer to produce BODIPY(.) (+) -azaBODIPY-C60 (.) (-) as major photochemical events. The charge-separated state persisted for few nanoseconds prior populating (3) C60 *, which in turn revealed an unusual triplet-triplet energy transfer to produce (3) azaBODIPY* prior returning to the ground state. These findings delineate the importance of multimodular systems in energy harvesting, and more importantly, their utility in building multifunction performing optoelectronic devices.

Ovarian tissue vitrification is more efficient than slow freezing in protecting oocyte and granulosa cell DNA integrity.

Ovarian tissue cryopreservation is the primary treatment modality currently available to women at risk of losing their ovarian function due to cytotoxic therapy. However, the impact of these techniques on the oocyte DNA integrity is not elucidated. Here we have investigated the effect of vitrification and conventional slow freezing of eight week old Swiss albino mouse ovarian tissues on the oocyte and granulosa cell DNA integrity using the comet assay. The intracellular levels of reactive oxygen species in oocytes was measured by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. The cryopreservation of ovarian tissue by the slow freezing technique resulted in a significantly higher level of DNA fragmentation in oocytes in comparison to vitrification (p < 0.05) whereas DNA fragmentation in granulosa cells was significantly higher than the control (p < 0.01). Further, reactive oxygen species were significantly elevated in oocytes derived from slow freezing when compared to vitrification (p < 0.05). Therefore, we conclude that the ovarian tissue slow freeze-thawing makes the oocyte and granulosa cells more vulnerable to DNA damage whereas vitrification appears to be a safer method than slow freezing for ovarian tissue cryopreservation.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential.

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential.

Germ cell abnormalities in streptozotocin induced diabetic mice do not correlate with blood glucose level.

PURPOSE: To assess the effect of streptozotocin induced hyperglycemia on germ cell integrity, DNA ploidy and methylation status for a period of two spermatogenesis cycles in adult male Swiss albino mice. METHODS: Streptozotocin injected mice were monitored for hyperglycemia at a regular interval for a period of 36 and 72 days. The DNA integrity in epididymal spermatozoa was determined by the comet assay. Flow cytometric analysis was done in germ cells to assess the DNA ploidy. The global methylation analysis in germ cells was done by 5-methyl cytosine immunostaining. RESULTS: Streptozotocin administration successfully resulted in hyperglycemic response which significantly affected serum testosterone level, sperm DNA integrity and DNA ploidy at the end of 36 days. However, no changes were observed in either epididymal sperm concentration or germ cell methylation status. In contrast, at the end of 76 days, although serum testosterone level, sperm DNA integrity and DNA ploidy status were unperturbed significantly in hyperglycemic group, the epididymal sperm concentration and methylation status of preleptotene/zygotene cells were significantly altered. Importantly, an attempt to find out the association between the blood glucose levels and the abnormalities in hyperglycemic group failed to demonstrate any correlation. CONCLUSIONS: The germ cell abnormalities observed in hyperglycemic group could be interpreted as a primary effect of streptozotocin and not due to hyperglycemia. Our results call for further evaluation of streptozotocin before its application to study the hyperglycemic responses on male germ cells.

Effects of apoA-V on HDL and VLDL metabolism in APOC3 transgenic mice.

Apolipoprotein A-V (apoA-V) and apoC-III are exchangeable constituents of VLDL and HDL. ApoA-V counteracts the effect of apoC-III on triglyceride (TG) metabolism with poorly defined mechanisms. To better understand the effects of apoA-V on TG and cholesterol metabolism, we delivered apoA-V cDNA into livers of hypertriglyceridemic APOC3 transgenic mice by adenovirus-mediated gene transfer. In response to hepatic apoA-V production, plasma TG levels were reduced significantly as a result of enhanced VLDL catabolism without alternations in VLDL production. This effect was associated with reduced apoC-III content in VLDL. Increased apoA-V production also resulted in decreased apoC-III and increased apoA-I content in HDL. Furthermore, apoA-V-enriched HDL was associated with enhanced LCAT activity and increased cholesterol efflux. This effect, along with apoE enrichment in HDL, contributed to HDL core expansion and alpha-HDL formation, accounting for significant increases in both the number and size of HDL particles. As a result, apoA-V-treated APOC3 transgenic mice exhibited decreased VLDL-cholesterol and increased HDL-cholesterol levels. ApoA-V-mediated reduction of apoC-III content in VLDL represents an important mechanism by which apoA-V acts to ameliorate hypertriglyceridemia in adult APOC3 transgenic mice. In addition, increased apoA-V levels accounted for cholesterol redistribution from VLDL to larger HDL particles. These data suggest that in addition to its TG-lowering effect, apoA-V plays a significant role in modulating HDL maturation and cholesterol metabolism.

Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation.

Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3',5'-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.