Dissociating Mechanisms That Underlie Seasonal and Developmental Programs for the Neuroendocrine Control of Physiology in Birds.

Long-term programmed rheostatic changes in physiology are essential for animal fitness. Hypothalamic nuclei and the pituitary gland govern key developmental and seasonal transitions in reproduction. The aim of this study was to identify the molecular substrates that are common and unique to developmental and seasonal timing. Adult and juvenile quail were collected from reproductively mature and immature states, and key molecular targets were examined in the mediobasal hypothalamus (MBH) and pituitary gland. qRT-PCR assays established deiodinase type 2 (DIO2) and type 3 (DIO3) expression in adults changed with photoperiod manipulations. However, DIO2 and DIO3 remain constitutively expressed in juveniles. Pituitary gland transcriptome analyses established that 340 transcripts were differentially expressed across seasonal photoperiod programs and 1,189 transcripts displayed age-dependent variation in expression. Prolactin (PRL) and follicle-stimulating hormone subunit beta (FSHß) are molecular markers of seasonal programs and are significantly upregulated in long photoperiod conditions. Growth hormone expression was significantly upregulated in juvenile quail, regardless of photoperiodic condition. These findings indicate that a level of cell autonomy in the pituitary gland governs seasonal and developmental programs in physiology. Overall, this paper yields novel insights into the molecular mechanisms that govern developmental programs and adult brain plasticity.

Photoperiodic physiology of summer breeding birds and a search for the role of eye.

Photoperiod regulation of gonadal cycles is well studied and documented in both birds and mammals. Change in photoperiod is considered as the most effective and important cue to time the initiation of the annual physiological cycles in birds. Approaching of long days (as observed in summer months), signal long-day breeding birds to initiation reproduction and other related functions. Birds and other non-mammalian vertebrates use the extraocular photoreceptors which may be present in the mediobasal hypothalamus (MBH) or associated regions to measure the photoperiodic time and so are different from mammals where only the eyes are lone photoreceptive organs. The downstream signaling involves thyroid responsive genes playing a crucial role in mediating photoperiodic signals in both birds and mammals. Role of eyes in the avian seasonal cycle has been a questionable issue with evidences both favoring and negating any role. We propose that morphological as well as physiological data argue that retinal photoreceptors can participate in gonadal cycle, at least in the quail and duck. The present review details the studies of photoneuroendocrine control of gonadal axis in birds and review evidences to decipher the role eyes in photoperiodic mediated physiologies in birds.

Luteal Phase Stimulation in the Same Cycle Is an Effective Strategy to Rescue POSEIDON Poor Responders with No Embryos after the First Follicular Stimulation.

Background: Poor responders may benefit from recruiting a 'second wave' of antral follicles within the same cycle. This concept forms the basis of double stimulation which has been named as 'DuoStim'. This protocol involves ovarian stimulation in both follicular and luteal phases with egg retrieval in each phase, respectively, to increase the number of oocytes and embryos in one menstrual cycle. This can be considered a potentially valuable option for women with poor ovarian reserve/response to maximise the number of oocytes retrieved in a single ovarian cycle in the shortest possible time. Aims: The aim of this study was to evaluate the efficacy of the DuoStim protocol in women classified as POSEIDON poor responders undergoing in vitro fertilization by comparing the embryological outcomes between the follicular and luteal phase stimulations in the same menstrual cycle. Settings and Design: This was a retrospective cohort study of 131 patients who enrolled to undergo DuoStim cycles from January 2021 to Sept. 2022, at a IVF center in a tertiary care hospital. Materials and Methods: The follicular phase stimulation used a standard antagonist protocol for the first oocyte retrieval. Thereafter, the luteal phase stimulation was started 3 days after the first retrieval, with the same dose of gonadotropin along with a daily 10 mg medroxyprogesterone acetate tablet, followed by a second oocyte retrieval. Blastocysts produced in both the phases were subsequently vitrified. Statistical Analysis Used: The paired t-test was used for comparing means and 95% confidence intervals (CIs) for different parameters. McNemar's test was used to compare paired proportions. The analysis was conducted using R statistical environment 4.2. Results: The mean number of oocytes retrieved and the mean number of utilizable blastocysts frozen per stimulation cycle were found to be significantly higher in the luteal phase as compared to the follicular phase (5.71 ± 3.95 vs. 4.87 ± 2.79, P = 0.02, and 1.43 ± 1.22 vs. 0.95 ± 1, P = 0.001, respectively). However, the mean fertilization rate and the mean blastocyst utilization rate were found to be similar between both the phases. The length of stimulation was found to be approximately 3 days longer in the luteal phase (12.63 ± 2.43 vs. 9.75 ± 1.85, P = 0.001). Overall, the odds of obtaining a usable blastocyst in the luteal phase was found to be significantly higher than in the paired follicular phase (73.9% vs. 57.7%, P = 0.012, odds ratio: 2.286 [95% CI: 1.186-4.636]). Also importantly, the luteal phase stimulation was able to rescue 68% (32/47) of patients where no blastocysts were formed in the follicular phase. Conclusion: Our data demonstrate that in women with poor reserve, the addition of luteal stimulation could increase the chances of achieving a pregnancy by significantly increasing the number of eggs and transferable embryos per menstrual cycle compared to follicular stimulation alone. Furthermore, luteal phase stimulation in the same cycle proved to be an effective strategy to rescue POSEIDON poor responders with no embryos after the first stimulation.

FSHß links photoperiodic signaling to seasonal reproduction in Japanese quail.

Annual cycles in daylength provide an initial predictive environmental cue that plants and animals use to time seasonal biology. Seasonal changes in photoperiodic information acts to entrain endogenous programs in physiology to optimize an animal's fitness. Attempts to identify the neural and molecular substrates of photoperiodic time measurement in birds have, to date, focused on blunt changes in light exposure during a restricted period of photoinducibility. The objectives of these studies were first to characterize a molecular seasonal clock in Japanese quail and second, to identify the key transcripts involved in endogenously generated interval timing that underlies photosensitivity in birds. We hypothesized that the mediobasal hypothalamus (MBH) provides the neuroendocrine control of photoperiod-induced changes in reproductive physiology, and that the pars distalis of the pituitary gland contains an endogenous internal timer for the short photoperiod-dependent development of reproductive photosensitivity. Here, we report distinct seasonal waveforms of transcript expression in the MBH, and pituitary gland and discovered the patterns were not synchronized across tissues. Follicle-stimulating hormone-ß (FSHß) expression increased during the simulated spring equinox, prior to photoinduced increases in prolactin, thyrotropin-stimulating hormone-ß, and testicular growth. Diurnal analyses of transcript expression showed sustained elevated levels of FSHß under conditions of the spring equinox, compared to autumnal equinox, short (<12L) and long (>12L) photoperiods. FSHß expression increased in quail held in non-stimulatory short photoperiod, indicative of the initiation of an endogenously programmed interval timer. These data identify that FSHß establishes a state of photosensitivity for the external coincidence timing of seasonal physiology. The independent regulation of FSHß expression provides an alternative pathway through which other supplementary environmental cues, such as temperature, can fine tune seasonal reproductive maturation and involution.

Deep Inception-ResNet: A Novel Approach for Personalized Prediction of Cumulative Pregnancy Outcomes in Vitro Fertilization Treatment (IVF).

Background: Infertility is one of the major causes of socioeconomic stress worldwide due to social stigma and stressful lifestyles. Despite technological advances, couples still undergo several IVF cycles for conceiving without knowing their true prognosis which is causing a huge social and medical impact, and the live birth rate continues to be relatively low (~ 25%). A prediction model that predicts IVF prognosis accurately considering the pre-treatment parameters before starting the IVF cycle will help clinicians and patients to make better-informed choices. Methods: In this study, clinical details of 2268 patients with 79 features who underwent IVF/ICSI procedure from January 2018 to December 2020, at the Center of IVF and Human Reproduction, Sir Ganga Ram Hospital were retrospectively collected. The machine learning model was developed considering features such as maternal age, number of IVF cycle, type of infertility, duration of infertility, AMH, indication for IVF, sperm type, BMI, embryo transfer, and ß-hCG value at the end of a fresh cycle and/or one subsequent frozen embryo transfer cycle was selected as the measure of outcome. Results: Compared to other classifiers, for an 80:20 train-test split with feature selection, the proposed Deep Inception-Residual Network architecture-based neural network gave the best accuracy (76%) and ROC-AUC score of 0.80. For tabular datasets, the applied approach has remained unexplored in previously made studies for reproductive health. Conclusion: This model is the starting point for providing a personalized prediction of a successful outcome for an infertile couple before they enter the IVF procedure. Supplementary Information: The online version contains supplementary material available at 10.1007/s13224-023-01773-9.

Low serum progesterone on day of transfer adversely impacts ongoing pregnancy rates in hormonally prepared single blastocyst frozen embryo transfer cycles.

OBJECTIVE: To evaluate if serum progesterone (P) levels on the day of transfer influence ongoing pregnancy rate (OPR) in hormonally prepared single blastocyst frozen embryo transfer (FET) cycles? STUDY DESIGN: Single center prospective cohort study conducted between June 2021 and August 2022 analyzed 217 single good quality blastocyst FET cycles hormonally prepared with oral estradiol valerate and micronized vaginal progesterone 400 mg twice daily. RESULTS: Mean serum P on the day of embryo transfer (ET) was 9.76 ± 5.19 ng/ml. Receiver operator curve (ROC) showed a significant predictive value of serum P levels on the day of ET for OPR, with an area under curve (AUC) (95 %CI) = 0.58 (0.49-0.66). Optimal serum P threshold for OPR was 7.7 ng/ml (Sensitivity 76.8%, Specificity 43.7%). 35.9% patients had serum P below this threshold. BMI was significantly higher (26.8 ± 3.7 vs 25.6 ± 4.3; p = 0.048) in patients with serum P < 7.7 ng/ml vs ≥ 7.7 ng/ml. OPR was significantly lower (24.4% vs 45.3%; p = 0.002) and clinical miscarriage rates significantly higher (37.9% vs 19.2%; p = 0.042) if serum P < 7.72 ng/ml vs ≥ 7.7 ng/ml. CONCLUSION: This study found that serum P level on the day of transfer in hormonally prepared FET cycles was a significant predictor of OPR.

Unraveling the Role of Thyroid Hormones in Seasonal Neuroplasticity in European Starlings (Sturnus vulgaris).

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.

Photoperiodic regulation of avian physiology: From external coincidence to seasonal reproduction.

Seasonal cycles of environmental cues generate variation in the timing of life-history transition events across taxa. It is through the entrainment of internal, endogenous rhythms of organisms to these external, exogenous rhythms in environment, such as cycling temperature and daylight, by which organisms can regulate and time life history transitions. Here, we review the current understanding of how photoperiod both stimulates and terminates seasonal reproduction in birds. The review describes the role of external coincidence timing, the process by which photoperiod is proposed to stimulate reproductive development. Then, the molecular basis of light detection and the photoperiodic regulation of neuroendocrine timing of seasonal reproduction in birds is presented. Current data indicates that vertebrate ancient opsin is the predominant photoreceptor for light detection by the hypothalamus, compared to neuropsin and rhodopsin. The review then connects light detection to well-characterized hypothalamic and pituitary gland molecules involved in the photoperiodic regulation of reproduction. In birds, Gonadotropin-releasing hormone synthesis and release are controlled by photoperiodic cues via thyrotropin-stimulating hormone-ß (TSHß) independent and dependent pathways, respectively. The review then highlights the role of D-box and E-box binding motifs in the promoter regions of photoperiodic genes, in particular Eyes-absent 3, as the key link between circadian clock function and photoperiodic time measurement. Based on the available evidence, the review proposes that at least two molecular programs form the basis for external coincidence timing in birds: photoperiodic responsiveness by TSHß pathways and endogenous internal timing by gonadotropin synthesis.

Neural programming of seasonal physiology in birds and mammals: A modular perspective.

Most animals in the temperate zone exhibit robust seasonal rhythms in neuroendocrine, physiological and behavioral processes. The integration of predictive and supplementary environmental cues (e.g., nutrients) involves a series of discrete, and interconnected brain regions that span hypothalamic, thalamic, mesencephalic, and limbic regions. Species-specific adaptive changes in these neuroendocrine structures and cellular plasticity have likely evolved to support seasonal life-history transitions. Despite significant advances in our understanding of ecological responses to predictive and supplementary environmental cues, there remains a paucity of literature on how these diverse cues impact the underlying neural and cellular substrates. To date, most scientific approach has focused on neuroendocrine responses to annual changes in daylength, referred to as photoperiod, due to the robust physiological changes to light manipulations in laboratory settings. In this review, we highlight the relatively few animal models that have been effectively used to investigate how predictive day lengths, and supplementary cues are integrated across hypothalamic nuclei, and discuss key findings of how seasonal rhythms in physiology are governed by adaptive neuroendocrine changes. We discuss how specific brain regions integrate environmental cues to form a complex multiunit or 'modular' system that has evolved to optimize the timing of seasonal physiology. Overall, the review aims to highlight the existence of a modular network of neural regions that independently contribute to timing seasonal physiology. This paper proposes that a multi-modular neuroendocrine system has evolved in which independent neural 'units' operate to support species-specific seasonal rhythms.

Natural cycle versus hormone replacement cycle for transferring vitrified-warmed embryos in eumenorrhoeic women. A retrospective cohort study.

OBJECTIVE: To compare pregnancy, miscarriage and live birth rates and cycle monitoring parameters between Natural Cycle (NC-FET) and Hormone replacement cycle (HRC-FET) in eumenorrhoeic women undergoing vitrified-warmed autologous embryo transfer. STUDY DESIGN: Single-centre retrospective cohort study analyzed 173 NC-FET and 507 HRC-FET cycles with transfer of day2/3/5/6 embryos. Natural cycle monitoring occurred with serial ultrasound with the first day of the scan determined by the shortest cycle frequency. Serum progesterone was ordered when ultrasound was ambiguous in ascertaining ovulation. For HRC-FET oral estradiol valerate was used in fixed or escalating doses with maximum daily dose of 12 mg. Transdermal estradiol gel was added when desired endometrial thickness was not achieved. Vaginal progesterone was introduced with Endometrial thickness(ET)> = 7 mm. Embryos were transferred after stage-appropriate progesterone exposure. Luteal support was given with vaginal progesterone in NC-FET and vaginal and oral progesterone in HRC-FET. Primary outcome was live-birth-rate. Secondary outcomes were ET, length-of-estrogenic-phase, numbers-of-ultrasounds&hormone-monitoring, pregnancy&miscarriage rate. The odds ratio for live-birth was adjusted for age, embryo number, previous-live-births, previous-losses, past-negative-ET-cycles, IVF-indication and embryo-developmental-stage. Quantitative variables were compared using unpaired-t-test and qualitative variables with chi-square test. Two tailed p-value < 0.05 was considered significant. Binary logistic regression was used to calculate adjusted odds for live-births. RESULTS: The two cohorts were comparable in age, infertility-duration, previous-live-births, previous-losses, past-negative-ET-cycles, IVF-indication and embryo-developmental-stage. Length-of-estrogenic-phase was significantly shorter for NC-FET than HRC-FET 14.32 ± 2.83vs.18.18 ± 4.48; p = 0.0001) as was mean ultrasound-monitoring-scans (2.73 ± 0.95vs. 3.3 ± 1.04; p = 0.0001). Mean-endometrial-thickness (8.75 ± 1.83vs. 8.5 ± 1.25; p = 0.25) and mean-hormonal-tests (1.75 ± 1.28 vs. 1.88 ± 0.69; p = 0.09) did not differ significantly between NC-FET vs HRC-FET. Significantly higher live births took place in NC-FET vs. HRC-FET (87/173 = 50.3%vs.204/507 = 40.2%;p = 0.026). No significant difference was found in pregnancy rate (66.5% vs. 58%; p = 0.058) or in the pregnancy loss rate (24.3%vs30.6%; p = 0.23). The odds ratio for live-births adjusted for relevant variables was 1.48 (1.03-2.13) in NC-FET compared to HRC-FET. CONCLUSIONS: NC-FET is a superior method of endometrial preparation compared to HRC-FET in eumenorrhoeic women since it has a shorter estrogenic phase, reduces patient visits to the hospital and improves live birth rates. Future adequately powered studies should look at antenatal and perinatal outcomes, patient satisfaction rates and cost-effectiveness in the two endometrial preparation regimes.

DNA Methylation Regulates Transcription Factor-Specific Neurodevelopmental but Not Sexually Dimorphic Gene Expression Dynamics in Zebra Finch Telencephalon.

Song learning in zebra finches (Taeniopygia guttata) is a prototypical example of a complex learned behavior, yet knowledge of the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA-sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age-dependent gene expression was found for various transcription factors (i.e., OTX2, AR, and FOS) involved in neurodevelopment. Incomplete dosage compensation, independent of DNA methylation, was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.

Indian Society for Assisted Reproduction Consensus Guidelines on Preimplantation Genetic Testing in In vitro Fertilization Clinics.

STUDY QUESTION: What are the good practice guidelines for Pre implantation genetic testing applicable in INDIA? WHAT IS ALREADY KNOWN: Pre-Implantation Genetic Testing (PGT) is not new in India. It is used to identify euploid embryos for transfer, thus enabling couples to achieve a healthy pregnancy. There has been a lot of controversy surrounding PGT in the international forums; most of these debates have failed to reach a consensus on whether PGT should be offered or its concerns be validated more. STUDY DESIGN SIZE DURATION: This is the report of a 2-day consensus meeting where two moderators were assigned to a group of experts to collate information on Pre implantation genetic testing and embryo biopsy practices in INDIA. This meeting utilised surveys, available scientific evidence and personal laboratory experience into various presentations by experts on pre-decided specific topics. PARTICIPANTS/MATERIALS SETTING METHODS: Expert professionals from ISAR representing clinical, embryological and genetic fields. MAIN RESULTS AND THE ROLE OF CHANCE: The report is divided into various components defining the terminologies, the various requirements, qualifications, recommendations on PGT -A,M,SR, and quality management: the report and recommendations of the expert panel reflect the discussion on each of the topics and try to lay down good practice points for labs to follow. LIMITATIONS REASONS FOR CAUTION: The recommendations are solely based on expert opinion. Future availability of data may warrant an update of the same. WIDER IMPLICATIONS OF THE FINDINGS: These guidelines can help labs across the country to standardise their PGT services and improve clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: The consensus meeting and writing of the paper was supported by funds from CooperSurgical India.

Hypothalamic plasticity in response to changes in photoperiod and food quality: An adaptation to support pre-migratory fattening in songbirds?

In latitudinal avian migrants, increasing photoperiods induce fat deposition and body mass increase, and subsequent night-time migratory restlessness in captive birds, but the underlying mechanisms remain poorly understood. We hypothesized that an enhanced hypothalamic neuronal plasticity was associated with the photostimulated spring migration phenotype. We tested this idea in adult migratory red-headed buntings (Emberiza bruniceps), as compared with resident Indian weaverbirds (Ploceus philippinus). Birds were exposed to a stimulatory long photoperiod (14L:10D, LP), while controls were kept on a short photoperiod (10L:14D, SP). Under both photoperiods, one half of birds also received a high calorie, protein- and fat-rich diet (SP-R, LP-R) while the other half stayed on the normal diet (SP-N, LP-N). Thirty days later, as expected, the LP had induced multiple changes in the behaviour and physiology in migratory buntings. Photostimulated buntings also developed a preference for the rich food diet. Most interestingly, the LP and the rich diet, both separately and in association, increased neurogenesis in the mediobasal hypothalamus (MBH), as measured by an increased number of cells immunoreactive for doublecortin (DCX), a marker of recently born neurons, in buntings, but not weaverbirds. This neurogenesis was associated with an increased density of fibres immunoreactive for the orexigenic neuropeptide Y (NPY). This hypothalamic plasticity observed in a migratory, but not in a non-migratory, species in response to photoperiod and food quality might represent an adaptation to the pre-migratory fattening, as required to support the extensive energy expenses that incur during the migratory flight.

Cholinergic Modulation of the Default Mode Like Network in Rats.

The discovery of the default mode network (DMN), a large-scale brain network that is suppressed during attention-demanding tasks, had major impact in neuroscience. This network exhibits an antagonistic relationship with attention-related networks. A better understanding of the processes underlying modulation of DMN is imperative, as this network is compromised in several neurological diseases. Cholinergic neuromodulation is one of the major regulatory networks for attention, and studies suggest a role in regulation of the DMN. In this study, we unilaterally activated the right basal forebrain cholinergic neurons and observed decreased right intra-hemispheric and interhemispheric FC in the default mode like network (DMLN). Our findings provide critical insights into the interplay between cholinergic neuromodulation and DMLN, demonstrate that differential effects can be exerted between the two hemispheres by unilateral stimulation, and open windows for further studies involving directed modulations of DMN in treatments for diseases demonstrating compromised DMN activity.

Molecular correlates of hypothalamic development in songbird ontogeny in comparison with the telencephalon.

Development of the songbird brain provides an excellent experimental model for understanding the regulation of sex differences in ontogeny. Considering the regulatory role of the hypothalamus in endocrine, in particular reproductive, physiology, we measured the structural (volume) and molecular correlates of hypothalamic development during ontogeny of male and female zebra finches. We quantified by relative quantitative polymerase chain reaction (rqPCR) the expression of 14 genes related to thyroid and steroid hormones actions as well as 12 genes related to brain plasticity at four specific time points during ontogeny and compared these expression patterns with the expression of the same genes as detected by transcriptomics in the telencephalon. These two different methodological approaches detected specific changes with age and demonstrated that in a substantial number of cases changes observed in both brain regions are nearly identical. Other genes however had a tissue-specific developmental pattern. Sex differences or interactions of sex by age were detected in the expression of a subset of genes, more in hypothalamus than telencephalon. These results correlate with multiple known aspects of the developmental and reproductive physiology but also raise a number of new functional questions.

Relationship Between Morphology, Euploidy and Implantation Potential of Cleavage and Blastocyst Stage Embryos.

AIM: The aim of this study was to investigate the relationship between the morphology, euploidy and implantation rate of cleavage stage and blastocyst stage embryos. SETTING: Institution-based, tertiary care in-vitro fertilization centre. STUDY DESIGN: This study included a retrospective data analysis of 306 embryos: 154 cleavage stage embryos and 152 blastocysts that underwent biopsy on day 3 and day 5/6, respectively, which were subsequently screened for aneuploidy by array comparative genomic hybridization analysis. MATERIALS AND METHODS: Both cleavage stage and blastocyst stage embryos were categorized according to their morphology into the following three groups: good, average and poor. In addition, blastocysts were categorized into day 5 and day 6 embryos on the basis of their developmental rate. RESULTS: The euploidy rate was found to be significantly higher for blastocysts with good morphology as compared to those with poor morphology, with 73.2, 50 and 40.5% euploid embryos in the good, average and poor morphology groups, respectively (P = 0.001). No significant association was found between day 3 embryo morphology and euploidy rates with 40.6, 29.3 and 25.8% euploid embryos in the three groups, respectively (P = 0.254). The implantation rates, as per morphology, for the transferred euploid cleavage stage and blastocyst stage embryos were 43.8, 37.5 and 0% (P = 0.354) and 51.7, 71.4 and 66.7% (P = 0.562) in the good, average and poor morphology groups, respectively. The euploidy rate for day 5 blastocysts was significantly higher (70% vs. 34.1%, P < 0.001) than that of day 6 blastocysts, but the implantation rate was similar in both the groups (58.8 and 50%, respectively). The miscarriage rates for the euploid cleavage stage and the blastocysts stage embryos were 18.2 and 8.3% (P = 0.575), respectively. CONCLUSION: Blastocyst morphology and the rate of development were found to be significantly associated with euploidy, whereas cleavage stage morphology was not. The implantation rates of the good quality, euploid cleavage stage embryos were higher than that of the poor quality embryos. The implantation rates were similar for all transferred euploid blastocysts, irrespective of their morphology or the rate of development.

Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential-as well as conditionally essential-regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions. In this chapter, we describe a recently modified experimental protocol used in our laboratory for TnSeq in the major human pathogen, Mycobacterium tuberculosis, as well as the related non-pathogenic mycobacterium, M. smegmatis. The method, which was developed in close consultation with pioneers in the field of mycobacterial genetics, includes the steps involved in preparing a phage stock, generating a mutant library, selection of the library under a specific experimental condition, isolation of genomic DNA from the pooled population of mutants, amplification of the sites of Tn insertion and, finally, determining the essential genomic regions by next-generation sequencing.

Preimplantation genetic screening for all 24 chromosomes by microarray comparative genomic hybridization significantly increases implantation rates and clinical pregnancy rates in patients undergoing in vitro fertilization with poor prognosis.

CONTEXT: A majority of human embryos produced in vitro are aneuploid, especially in couples undergoing in vitro fertilization (IVF) with poor prognosis. Preimplantation genetic screening (PGS) for all 24 chromosomes has the potential to select the most euploid embryos for transfer in such cases. AIM: To study the efficacy of PGS for all 24 chromosomes by microarray comparative genomic hybridization (array CGH) in Indian couples undergoing IVF cycles with poor prognosis. SETTINGS AND DESIGN: A retrospective, case-control study was undertaken in an institution-based tertiary care IVF center to compare the clinical outcomes of twenty patients, who underwent 21 PGS cycles with poor prognosis, with 128 non-PGS patients in the control group, with the same inclusion criterion as for the PGS group. MATERIALS AND METHODS: Single cells were obtained by laser-assisted embryo biopsy from day 3 embryos and subsequently analyzed by array CGH for all 24 chromosomes. Once the array CGH results were available on the morning of day 5, only chromosomally normal embryos that had progressed to blastocyst stage were transferred. RESULTS: The implantation rate and clinical pregnancy rate (PR) per transfer were found to be significantly higher in the PGS group than in the control group (63.2% vs. 26.2%, P = 0.001 and 73.3% vs. 36.7%, P = 0.006, respectively), while the multiple PRs sharply declined from 31.9% to 9.1% in the PGS group. CONCLUSIONS: In this pilot study, we have shown that PGS by array CGH can improve the clinical outcome in patients undergoing IVF with poor prognosis.

Bird eyes distinguish summer from winter: Retinal response to acute photoperiod change in the night-migratory redheaded bunting.

Eyes are the part of the circadian timekeeping system but not involved in the photoperiod regulated seasonal physiology in songbirds. Here, two experiments tested whether eyes detect and respond to seasonal change in the photoperiod environment, by examining gene and protein expressions in the retinas of redheaded buntings exposed to a single long day (LD, 16L:8D), with controls on short days (SD, 8L:16D). In the first experiment, mRNA expression of genes implicated in the light perception (opsins, rhodopsin, neuropsin, melanopsin, peropsin) and photoperiod induction (eya3, tsh-ß, dio2, dio3) was measured at hours 15 and 19 (hour 0 = light on) on the first long day. There was a significant increase in the eya3, tsh-ß and dio2 mRNA expression, albeit with a temporal difference, and decrease in the neuropsin mRNA expression in buntings on the first long day. There was no change in the dio3, rhodopsin, melanopsin and peropsin mRNA expressions on exposure to long days. The second experiment immunohistochemically examined the eya3, tsh-ß and rhodopsin peptide expressions. eya3 was expressed in both light conditions, but with a significant higher levels in the retinal photoreceptor layer (PRL) under LD, as compared to SD. Similarly, tsh-ß was expressed in the PRL of LD retinas only. Rhodopsin levels were not significantly different between SD and LD conditions, however. These results for the first time show photoperiod-dependent molecular switches in the bunting retina, similar to the well documented thyroid hormone response genes based molecular cascades in the avian hypothalamus.

Circadian synchronization determines critical day length for seasonal responses.

A photoperiodic species initiates fat deposition (in migrants) and gonadal recrudescence in response to a specific duration of natural daylight, called critical day length (CD), when light extends in the inductive phase of the endogenous circadian rhythm of photoinducibility (CRP). The molecular basis of species-specificCD, determined by the entrainment of the CRP, has been poorly understood. To investigate this, we measured expression levels of genes implicated in the photoperiod-induced changes in reproduction (EYA3, TSH beta, DIO2, DIO3, GNRH and GNIH) and metabolism (SIRT1, HMGCR, FASN and PPAR alpha) in photosensitive redheaded buntings subjected to light-dark cycles of varying period lengths (T-photocycles). Buntings were exposed to six T22, T24 or T26 photocycles, with 1h additional light at night falling at different phases of the entrained CRP (T2211L=6L:4D:1L:11D; T2411L=6L:4D:1L:13D,T2412L=6L:5D:1L:12D, T2413L=6L:6D:1L:11D; T2612L=6L:5D:1L:14D). Photoinduction at genetic and phenotypic levels in T2412L and T2413L, not T2411L, groups confirmed CD being close to 12h in buntings under T24. Compared to T24, exposure to T22 advanced CD by 1h, as evidenced by photoinduction in the T2211L, not T226L, group. Similarly, CD appeared to be delayed under T26, with no photoinduction in the T2612L group. Further, to show that induction of response under a T-photocycle was because of the interaction of inductive phase of the CRP with 1h during the dark period in each cycle, not with the 6h main light periods falling 2h earlier each successive 24hday in a T22 paradigm, a group of buntings was exposed to 6L:16D (T226L), to which they did not respond. The mRNA expression of genes, particularly TSH beta, DIO2, DIO3 and PPAR alpha, was significantly correlated with changes in reproductive and metabolic phenotypes. These results suggest CRP-entrainment based genetic regulation of the CD, and extend the idea that synchronization with environment is a critical measure in a seasonal species for its temporal adaptation in the wild.