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Objective: To explore the ability of diacyl glycerol (DAG) and inositol triphosphate (IP3), two major secondary messengers in the calcium signaling pathway, in activating oocytes. Material and Methods: Oocyte cumulus complex obtained from superovulated Swiss albino mice were incubated in M16 medium with liposome-encapsulated 1,2-Dipalmitoyl-sn-glycerol (LEDAG) and/or IP3 for 3 h. Strontium chloride was used as positive control. The activation potential, ploidy status, and blastocyst rate was calculated. Results: Both DAG and IP3, individually, induced activation in ~98% of oocytes, which was significantly higher (p<0.01) than activation induced by strontium chloride (60%). Delayed pronucleus formation and a higher percentage of diploid parthenotes was observed in oocytes activated with LEDAG and/or IP3. However, these embryos failed to progress beyond the 6-8-cell stage. Only when the medium was supplemented with LEDAG (5 μg/mL) and IP3 (10 μg/mL) could activated oocytes progress till the blastocyst stage (5.26%), which was lower than the blastocyst rate in the positive controls (13.91%). Conclusion: The results of the present study indicate that DAG and IP3 can induce delayed oocyte activation and poor development of parthenotes in vitro. [ABSTRACT FROM AUTHOR]

BACKGROUND: Oocyte retrieval in women with low ovarian reserve is challenging and often results in poor pregnancy outcomes. AIM: The aim was to compare outcomes of oocyte retrieval using three aspiration methods in women with normal and low antral follicle counts (AFCs). STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary care hospital. MATERIALS AND METHODS: Data from 172 women who underwent in-vitro fertilization/intracytoplasmic sperm injection over 3 years was studied after dividing into three groups: Group A (96) - normal AFC (≥10), direct oocyte retrieval at 120 mmHg, Group B (41) - low AFC (<10), direct retrieval at 140-mmHg, Group C (35) - low AFC, follicular flushing, and aspiration at 120-mmHg. In each group, oocyte yield, embryo quality, and pregnancy out comes were assessed. RESULTS: The oocyte and embryo yield in Groups A and B were comparable (P > 0.05) but were significantly less in Group C (P < 0.05). The pregnancy rates in Groups A and B were similar (35.4%, 39%) but were significantly less (17.1%) in Group C (P < 0.05). The live birth rate was 76% in Group A, 69% in Group B, and 50% in Group C. The miscarriage rates in the three groups were 21%, 31%, and 50%, respectively. CONCLUSION: Direct oocyte retrieval with 140-mmHg showed significantly better oocyte yield and pregnancy outcomes compared to flushing and aspiration in women with low AFC. [ABSTRACT FROM AUTHOR]

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 © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Objective: The aim of this experimental prospective study was to investigate the efficacy of single and combination sperm wash methods for their ability to isolate DNA intact spermatozoa. Material and Methods: Sperm DNA damage was introduced by local testicular irradiation in male mice and the extent of damage was quantified by comet assay. The spermatozoa were subjected to single (swim up or density gradient method) and also a combination of sperm wash techniques. The DNA integrity in various sub-fractions of wash techniques was evaluated. Results: The amount of DNA damaged sperm did not differ between individual fractions when single wash technique was applied. However, a combination of density gradient and swim-up techniques significantly reduced (p<0.01) the number of DNA damaged sperm in the final population. Conclusion: The combination of density gradient separation and swim-up method is effective in eliminating DNA damaged spermatozoa. [ABSTRACT FROM AUTHOR]

AIM: The present study evaluates the impact of controlled slow cooling and rapid freezing techniques on the sperm chromatin integrity in teratozoospermic and normozoospermic samples. SETTING: The study was done in a university infertility clinic, which is a tertiary healthcare center serving the general population. DESIGN: It was a prospective study designed in vitro. MATERIALS AND METHODS: Semen samples from normozoospermic (N=16) and teratozoospermic (N=13) infertile men were cryopreserved using controlled cooling and rapid freezing techniques. The sperm chromatin integrity was analyzed in fresh and frozen-thawed samples. STATISTICAL ANALYSIS USED: Data were reported as mean and standard error (mean ± SEM) of mean. The difference between two techniques was determined by a paired t-test. RESULTS: The freeze-thaw induced chromatin denaturation was significantly (P<0.01) elevated in the post-thaw samples of normozoospermic and teratozoospermic groups. Compared to rapid freezing, there was no difference in the number of red sperms (with DNA damage) by the controlled slow cooling method in both normozoospermic and teratozoospermic groups. Freeze-thaw induced sperm chromatin denaturation in teratozoospermic samples did not vary between controlled slow cooling and rapid freezing techniques. CONCLUSIONS: Since the controlled slow cooling technique involves the use of expensive instrument and is a time consuming protocol, rapid freezing can be a good alternative technique for teratozoospermic and normozoospermic samples when sperm DNA damage is a concern. [ABSTRACT FROM AUTHOR]

Objective: Sperm DNA damage is known to cause developmental failure and reduction in the numbers of live offspring and the effects of damages range as diverse as embryonic death and cancer susceptibtity in the offspring. Here we report the in vitro proliferation ability of the inner cell mass of the mouse embryos derived from the DNA damaged sperm and its association with post implantation developmental potential. Material and Method: Day 3.5 mouse embryos derived from the DNA damaged sperm were cultured on MEF feeder layer and proliferation ability of the inner cell mass was assessed for six days. The post implantation developmental competence was studied by fetoplacental analysis on day 18 of gestatlon. Result: The development of embryos derived from 6 Gy irradiated sperm demonstrated heterogeneous growth on day 3.5 as approximately 1/3rd of the embryos failed to undergo compaction and demonstrated high frequency of microouclei. In addition embryos showing developmental delay on day 3.5 failed to form any outgrowth during 6 day of in vitro culture. The fetoplacental analysis on day 18 of gestation showed a 50% reduction in the number of fetus derived from the DNA damaged sperm although the number of implantations was not affected. Conclusions: Our study demonstrates that DNA damage in sperm can lead to preimplantation embryonic developmental delay resulting in defective ICM proliferation possibly due to increased genomic instability and such embryos die in utero. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms, including DNA methylation and histone modifications, govern chromatin arrangement in sperm, enhancing motility and safeguarding DNA integrity for accurate epigenetic inheritance. Abnormal methylation is linked to poor sperm quality and fertility issues, underscoring the need to study sperm DNA methylation and its impact on sperm function and embryo development in assisted reproductive technology. In this study, processed spermatozoa from 75 normozoospermic and 15 abnormal ejaculates were examined for sperm global DNA methylation levels using a colourimetric absorbance method. Although semen characteristics were poor in abnormal ejaculates, no significant correlation was found between sperm global DNA methylation levels and sperm characteristics in either normozoospermic or abnormal cohorts. However, mean global DNA methylation levels were significantly lowered in abnormal sperm samples compared to normozoospermic samples (p < 0.05). Furthermore, injecting spermatozoa from these patients (N = 50) into donor oocytes did not show a significant relationship between sperm global DNA methylation and embryo developmental competence. These findings highlight the limitation of sperm global DNA methylation as a biomarker for embryo development and quality. [ABSTRACT FROM AUTHOR]

Fertility restoration potential of immature testicular tissue (ITT) depends on the number of spermatogonial cells in the retrieved tissue prior to cryopreservation in oncofertility programme. There are limited data on the association between type of malignancy and testicular germ cell population. Hence, this study is aimed to investigate the spermatogonial and Sertoli cell population in ITT retrieved from 14 pre-pubertal boys who opted for fertility preservation. Histopathological and immunochemical analysis of seminiferous tubules from haematological (N = 7) and non-haematological (N = 7) malignant patients revealed 3.43 ± 2.92 and 1.71 ± 1.81 spermatogonia per tubular cross section (S/T), respectively. The Sertoli cell number was comparable between haematological and non-haematological group (18.42 ± 3.78 and 22.03 ± 10.43). Spermatogonial quantity in ITT did not vary significantly between haematological and non-haematological cancers. This observation, though preliminary, would contribute to the limited literature on paediatric male oncofertility. [ABSTRACT FROM AUTHOR]

Cryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze–thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level (p < 0.05), and reduced apoptosis (p < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy. [ABSTRACT FROM AUTHOR]

Aim: To find out effect of serum luteinizing hormone (LH) levels on Day 1 (Day 2 of cycle) and Day 5 of stimulation and Day of trigger hCG in controlled ovulation stimulation with antagonist protocol of IVF-ICSI cycle on cycle outcome. Materials and Methods: This retrospective study was conducted in a University Hospital setting. One hundred and 62 patients underwent 165 cycles of controlled ovarian stimulation (COS) with recombinant Follicle stimulating hormone (rFSH) and Gonadotropin releasing hormone (GnRH) antagonist protocol in one year were included. Serum LH levels estimated on Day 1, Day 5 and the day of trigger hCG injections were divided into three groups based on the percentile and outcome measured. Results: The average number of follicles >18 mm in size were significant in the patients with LH levels between 25th to 75th percentile group on Day 1, Day 5 and Day of trigger hCG. The fertilization rate was significant (p= 0.04) in the patients with LH levels < 25thpercentile on the day of trigger hCG. Oocyte recovery rate, oocyte maturation rate and average number of best quality embryos (Grade 1) were not affected significantly in all three groups. Conclusion: In GnRH antagonist and rFSH protocols, low serum LH concentrations on the day of trigger hCG has better fertilization rate. LH levels between 25th and 75th percentile have an influence on the average number of > 18 mm size follicles. However, the LH level on Day 1, Day 5 and Day of hCG does not affect the cycle outcome in COS with antagonist protocol of IVF cycle. Hence, LH estimation is not mandatory in ART cycles with GnRH antagonist protocol. [ABSTRACT FROM AUTHOR]

OBJECTIVES: To evaluate the effectiveness of two different systems i.e. circulating water bath and carbon dioxide (CO2) incubator in extracting motile sperm for IUI programme and their effect on pregnancy outcome. METHODS: The study was performed on sixty-two patients recruited for ovulation induction followed by intrauterine insemination (IUI) in University fertility clinic. The patients were randomly divided into two groups and sperm preparation was performed with either water bath or CO2 incubator system. The efficiency of the two systems was analyzed in relation to pregnancy outcome. RESULTS: There was no significant difference in the efficacy of water bath and CO2 system with respect to the quality of sperm extracted and pregnancy outcome. Although pregnancy rate was marginally higher in water bath group, it was not statistically significant. CONCLUSION: CO2-free system can be a cost-effective approach in IUI programme which does not compromise with pregnancy rate. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To estimate acetylcholinesterase (AChE), protein thiols (PT), ceruloplasmin (CP) and C-reactive proteins (CRPs) to assess any change in their levels following intrauterine insemination (IUI). MATERIALS AND METHODS: Forty-two patients aged 31 ± 4.65 years (mean ± SD) with primary infertility selected for IUI. All of them had induced ovulation with clomiphene citrate 50 mg from day 2 to day 6. After taking the consent, 2 ml of blood was withdrawn before and after 24 h of IUI for biochemical estimations. RESULTS: We observed a significant decrease in plasma CP, PT and RBC AChE (P < 0.001) following IUI compared with the respective pre-procedure levels. Highly sensitive CRP showed a marginal increase after IUI. CONCLUSION: Fluctuations in levels of the above parameters point to their role in the female reproductive system and in the outcome of the IUI. [ABSTRACT FROM AUTHOR]

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and β3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv β3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The unique epigenetic architecture that sperm cells acquire during spermiogenesis by retaining <15% of either canonical or variant histone proteins in their genome is essential for normal embryogenesis. Whilst heterogeneous levels of retained histones are found in morphologically normal spermatozoa, their effect on reproductive outcomes is not fully understood. Methods: Processed spermatozoa (n = 62) were tested for DNA integrity by sperm chromatin dispersion assay, and retained histones were extracted and subjected to dot‐blot analysis. The impact of retained histone modifications in normozoospermic patients on sperm functional characteristics, embryo quality, metabolic signature in embryo spent culture medium and pregnancy outcome was studied. Results: Dot‐blot analysis showed heterogeneous levels of retained histones in the genome of normozoospermic ejaculates. Post‐wash sperm yield was affected by an increase in H3K27Me3 and H4K20Me3 levels in the sperm chromatin (p < 0.05). Also, spermatozoa with higher histone H3 retention had increased DNA damage (p < 0.05). Spermatozoa from these cohorts, when injected into donor oocytes, correlated to a significant decrease in the fertilisation rate with an increase in sperm histone H3 (p < 0.05) and H3K27Me3 (p < 0.01). An increase in histone H3 negatively affected embryo quality (p < 0.01) and clinical pregnancy outcome post‐embryo transfer (p < 0.05). On the other hand, spent culture medium metabolites assessed by high‐resolution (800 MHz) nuclear magnetic resonance showed an increased intensity of the amino acid methionine in the non‐pregnant group than in the pregnant group (p < 0.05) and a negative correlation with sperm histone H3 in the pregnant group (p < 0.05). Discussion and conclusion: Histone retention in spermatozoa can be one of the factors behind the development of idiopathic male infertility. Such spermatozoa may influence embryonic behaviour and thereby affect the success rate of assisted reproductive technology procedures. These results, although descriptive in nature, warrant further research to address the underlying mechanisms behind these clinically important observations. [ABSTRACT FROM AUTHOR]

Oocyte cryopreservation is offered to women of various age groups for both health and social reasons. Oocytes derived from either controlled ovarian stimulation or in vitro maturation (IVM) are cryopreserved via vitrification. As maternal age is a significant determinant of oocyte quality, there is limited data on the age-related susceptibility of oocytes to the vitrification-warming procedure alone or in conjunction with IVM. In the present study, metaphase II oocytes obtained from 2, 6, 9, and 12 month old Swiss albino mice either by superovulation or IVM were used. To understand the association between maternal age and oocyte cryotolerance, oocytes were subjected to vitrification-warming and compared to non vitrified sibling oocytes. Survived oocytes were evaluated for mitochondrial potential, spindle integrity, relative expression of spindle checkpoint protein transcripts, and DNA double-strand breaks. Maturation potential and vitrification-warming survival were significantly affected (p < 0.001 and p < 0.05, respectively) in ovulated oocytes from the advanced age group but not in IVM oocytes. Although vitrification-warming significantly increased spindle abnormalities in ovulated oocytes from advanced maternal age (p < 0.01), no significant changes were observed in IVM oocytes. Furthermore, Bub1 and Mad2 transcript levels were significantly higher in vitrified-warmed IVM oocytes (p < 0.05). In conclusion, advanced maternal age can have a negative impact on the cryosusceptibility of ovulated oocytes but not IVM oocytes in mice. [ABSTRACT FROM AUTHOR]