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

Oncofertility awareness among primary care physicians in India [version 1; peer review: 2 approved, 1 not approved]

Background: Primary care physicians not only coordinate referrals to oncology services but can play a crucial role in successful fertility preservation referrals in cancer-diagnosed patients. Hence, it is important to assess their knowledge and attitudes towards fertility preservation. Methods: An eighteen-item oncofertility survey was administered to primary care physicians between May 2019 to September 2020.  Results: A total of forty-six responses were received and analysed. About 60% of primary care physicians did not have adequate knowledge about available fertility preservation options and only 26-32% were aware of international guidelines recommending fertility preservation in cancer patients.  Conclusions: Imparting awareness and knowledge of fertility preservation and its options to primary care physicians could enable an integrated cancer care model while also facilitating successful oncofertility referrals in countries like India

Oncofertility awareness among primary care physicians in India [version 2; peer review: 2 approved, 1 not approved]

Background: Primary care physicians not only coordinate referrals to oncology services but can play a crucial role in successful fertility preservation referrals in cancer-diagnosed patients. Hence, it is important to assess their knowledge and attitudes towards fertility preservation. Methods: An eighteen-item oncofertility survey was administered to primary care physicians between May 2019 to September 2020.  Results: A total of forty-six responses were received and analysed. About 60% of primary care physicians did not have adequate knowledge about available fertility preservation options and only 26-32% were aware of international guidelines recommending fertility preservation in cancer patients.  Conclusions: Imparting awareness and knowledge of fertility preservation and its options to primary care physicians could enable an integrated cancer care model while also facilitating successful oncofertility referrals in countries like India

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

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

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. Copyright (C) 2012 John Wiley & Sons, Ltd

Proteinaceous sperm motility inhibitory factor from the female Indian garden lizard Calotes versicolor

Female sperm storage is an intriguing adaptation exhibited by a wide array of both vertebrates and invertebrates. The mechanisms underlying female sperm storage have remained elusive. Using the Indian garden lizard Calotes versicolor as a model organism, we investigated the role of low and high molecular weight factors in this phenomenon. Previously, we demonstrated three distinct phases of the reproductive cycle in this animal with live, motile spermatozoa recovered from the uterovaginal region during the reproductive phase. In the present study, we analysed the uterovaginal contents using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and identified an abundant protein band corresponding to ~55 kDa regardless of the phase of the reproductive cycle. Analysis of the purified protein by liquid chromatography–tandem mass spectrometry suggested a unique protein without any homology to the National Center for Biotechnology Information database. Exogenous addition of this protein to washed spermatozoa derived from the epididymis reversibly inhibited sperm motility in a concentration- and time-dependent manner, suggesting it plays a key role in sperm storage. These studies are likely to offer new avenues to unravel the secrets of female sperm storage seen across the animal taxa and may have novel applications not only in reproductive biology, but also in general cell storage and preserving endangered animal species

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

Mitochondrial distribution and activity as measured by Rhodamine 123 and JC1 staining of oocytes.

<p>A. Oocytes retrieved from superovulated mice, subjected to mock ICSI were assessed for mitochondrial distribution. The percentage of oocytes displaying uniform (closed bar); and aggregated (grey bar) distribution in standard control (N = 28), ICSI control (N = 49) and ICSI group (N = 36), was determined. B. <i>In vitro</i> matured metaphase II oocytes, subjected to mock ICSI were evaluated for mitochondrial distribution. The percentage of oocytes displaying uniform (closed bar); aggregated (grey bar) and peripheral (open bar) distribution in standard control (N = 33), ICSI control (N = 33) and ICSI group (N = 32), was determined. ^aP <0.05: Uniform distribution pattern in standard control of figure A Vs Standard control in figure B. ^bP < 0.001: Standard control Vs ICSI control. ^cP < 0.01: Standard control Vs ICSI group. ^dP < 0.0001: percentage of oocytes displaying peripheral distribution in ICSI group with other two groups. C. Mitochondrial activity as measured by the JC1 ratio in IVM oocytes in standard control (N = 81); ICSI control (N = 69) and ICSI group (N = 68). Please note that difference were not significant.</p