Study question Does sperm affect the implantation and early embryogenesis? Summary answer Deranged sperm genomic integrity, limited repair mechanisms, aberrant gene expression have the potential to be transmitted to developing embryo and affect implantation and embryonic development. What is known already The terminally differentiated, transcriptionally quiscent sperm cells are vulnerable to a host of factors which might affect its function. The spermatozoa with truncated repair mechanisms have the potential to fertilize the oocyte, but eventually result in high rates of pre and post implantation losses. Oxidative stress, deranged genomic integrity, aberrant gene expression in the sperm cell has the potential to affect fertilisation potential, implantation and embryonic viability. This has been seen to be correlated with decreased fertilisation, clevage rates and blastocyst development and thus impaired implantation rates. Study design, size, duration A case control study of 75 men from infertile couples who had recurrent implantation failure (RIF) in IVF cycles and 75 fertile controls at AIIMS, New Delhi, India. Study duration was 2 years. Participants/materials, setting, methods Semen samples from men from couples who had RIF were analyzed as per WHO 2010. Sperm reactive oxygen species (ROS) and DNA fragmentation index (DFI) was assessed by chemiluminiscence and sperm chromatin structure assay (SCSA) respectively. Relative sperm telomere length was evaluated from sperm DNA by q-PCR analysis. The expression of genes pertinent for early embryonic development was done by qPCR. The relative quantification was done after normalizing with GAPDH and β-actin by 2-ΔΔCt method. Main results and the role of chance Seminal ROS levels (RLU/sec/million sperm) were seen to be significantly higher [57.75 (10.1-1186.9)] in cases with respect to controls [16.7(1.15-53.9)] (p < 0.001***). The mean DFI of men undergoing ART was significantly higher (37.7 ± 5.7) vs 23.2 ± 4.6%; p < 0.001***) in cases as compared to controls. ROS and DFI levels correlated negatively with sperm concentration and progressive motility (p < 0.001***). We analyzed the expression of FOXG1, SOX3, STAT4, RPS6, RBM9, RPL10A, RPS17, RPL29, WNT5A, HSP90, TOMM7, EIF5A genes. The expression of SOX3, RBM9, WNT5A, HSP90, TOMM7, and EIF5A showed a significant difference from control levels. The relative sperm telomere length was found to be significantly lower in RIF patients as compared to controls (p < 0.001***). Limitations, reasons for caution The current study was a case control study and lacked randomization and also is limited by low sample size. There is a lack of stratification in the enrollment of participants due to difference in specific risk of occurrence and varied clinical history. Wider implications of the findings The analysis of spermatozoal gene expression is important for understanding the sperm differentiation, fertilization and early embryonic events. Correlation with oxidative stress, genomic integrity and telomere length may help in regulationof gene expression. It may help in establishing sperm gene expression as a potential biomarker. Trial registration number Not applicable

Study question Does adding gonadotropin-releasing hormone agonist (GnRHa) to hCG trigger increases the number of high-grade embryos in GnRH antagonist protocol in fresh non-donor IVF? Summary answer Final oocyte maturation triggered by dual trigger increases the number of MII oocytes thus transferring good-quality embryos and cryopreserving surplus embryos compared to hCG trigger. What is known already hCG has been conventionally used as a ‘faux’ LH surge to bring about final oocyte maturation due to structural similarity between the two. GnRH agonist, on the other hand, induces a more physiological gonadotropin surge for follicular maturation, but is associated with luteal phase deficiency. Recent studies have shown that combining GnRHa with hCG trigger improves oocyte maturation and embryo quality with the added benefit of a luteal phase support, thereby improving IVF outcomes in terms of both embryological and reproductive outcomes. Study design, size, duration A single-center, open labelled, randomized controlled trial including 100 normal responder patients between 21-38 years undergoing IVF using GnRH antagonist protocol between January 2020 to August 2021. The study excluded patients with the presence of other variables of adverse outcomes like diminished ovarian reserve (AFC < 5 or AMH < 1.2 ng/ml), endocrine disorders, thin endometrium (<6mm), previous history of uterine surgeries, and high responders. Participants/materials, setting, methods 100 patients undergoing fresh IVF cycle using GnRH antagonist protocol were randomized after informed consent to receive either dual trigger (Leuprolide acetate 1 mg + rhCG 250 mcg, n = 50) or single hCG trigger (rhCG 250 mcg, n = 50). Oocyte retrieval was done 35-37 hours after trigger followed by IVF/ICSI, as indicated. Oocyte and embryo grading was done using Istanbul consensus. Analysis was done by ITT. Outcomes were analyzed using Independent t-test and Chi-square test. Main results and the role of chance The baseline characteristics were comparable in both arms. the number of MII oocytes retrieved (7.82 versus 5.92, p = 0.003) and the number of day-3 grade-1 embryos (4.24 versus 1.8, p < 0.001) were higher in the dual trigger group, whereas fertilization rates between the two groups (91.82% versus 88.51%, p=NS) were comparable. Consequently, the number of embryos cryopreserved (2.68 versus 0.94, p < 0.001) were significantly higher in the dual trigger group. However, the implantation rate between the two groups (21% versus 19.6%, p = 0.770) was comparable. The serum LH levels 12 hours post trigger were measured in both the arms and as expected, high serum LH values were documented in the dual trigger group (46.23 mIU/ml vs 0.93 mIU/ml, p < 0.0001). Limitations, reasons for caution Due to the impact of the Covid-19 pandemic causing an intermittent pause in IVF services at our center, a smaller sample size of 100 patients could be enrolled in the study, and reproductive outcomes in terms of live births and cumulative live births could not be assessed Wider implications of the findings This study, though small, has contributed to some evidence of redesigning the dual trigger in all antagonist cycles, with the exception of high responders and PCOS patients. The addition of GnRHa to hCG trigger has led to the possibility of cryopreserving surplus embryos thereby increasing the cumulative live births. Trial registration number CTRI/2020/08/027030

Study question Does intra-ovarian instillation of platelet rich plasma (PRP) improves the clinical outcome of IVF cycles in women with Diminished Ovarian reserve (DOR)? Summary answer PRP instillation leads to consistent improvement in Antral follicle count (AFC), thus achieving clinical pregnancy rate of 33.3% per cycle in women with DOR. What is known already There is rising incidence of females with diminished ovarian reserve (DOR) especially among Asian ethnicity. With the emergence of regenerative medicine, multiple studies have evaluated the role of intra-ovarian PRP, demonstrating a beneficial role in improving ovarian reserve parameters (serum Follicular stimulating hormone (FSH), serum anti-Mullerian hormone (AMH), AFC). Despite its’ favorable effects on biochemical markers and AFC, data regarding improvement in clinical outcome remains elusive and led to inception of this study. Study design, size, duration A prospective interventional study was conducted at Division of Reproductive Medicine of a tertiary care institute. 41 infertile females aged 20-39 years with DOR (AMH <1.2 ng /ml; AFC<5) were enrolled in the study during a 6-month period beginning from August 2021. Participants/materials, setting, methods After informed consent, patients received fresh autologous PRP, prepared from 30 ml venous blood. 1.5ml of PRP instilled in each ovarian stroma between day 7-10 of menstrual cycle under sedation. Patients were followed up for three-consecutive months to assess ovarian reserve parameters including serum FSH, AMH and AFC. Patients showing significant improvement in parameters were recruited for fresh IVF cycles using Antagonist protocol with 1% transdermal testosterone. Outcomes were analysed using linear mix effect model. Main results and the role of chance The average platelet concentration in PRP was ∼10,00,000 platelets/µL. The mean age of enrolled patients was 31.22±4.16 years. Linear improvement in AFC (3.63 vs 6.98 vs 7.97 vs 6.90, p<0.001) was observed from baseline to three consecutive follow-up months with maximal response witnessed in second month in 57.1% of those undergoing IVF cycle. However, there was no significant difference in Serum FSH (p=0.11) and AMH (p=0.16) from the baseline post intervention. Of the 41 patients, 35 (85.3%) responded to the treatment and underwent IVF antagonist cycle. 5 out of 35 IVF cycles were cancelled mid-cycle due to poor ovarian response. The mean dose of gonadotropin requirement was 2667.5±281.1 IU (Follicular stimulating hormone) and 1400±337.3 IU (Human menopausal gonadotropin). The average number of oocytes retrieved was 5.7±2.2 whereas mean number of MII oocytes was 4.63±1.85. The fertilization rate and the cleavage rate were 92.4% and 74.1% respectively. Of the thirty patients, eight patients underwent day 2 transfer due to poor grade of embryos. Mean number of grade 1- day 3 embryos was 1.25±0.55 with surplus embryos available for cryopreservation in 14 patients. The overall clinical pregnancy rate per transfer was 33.33%. No adverse events were reported. Limitations, reasons for caution This was a prospective single arm study. A randomized controlled trial comprising a “no-treatment” arm would establish a Level-I evidence. However, “no-treatment” arm in a developing country like ours, imposes financial burden on the couple with no guaranteed clinical success and thus raising ethical concern and need for ovarian rejuvenation. Wider implications of the findings With the impetus to provide a biological child to these DOR women, intra-ovarian PRP instillation as a method of ovarian rejuvenation holds promising results. Evidently, PRP is not only effective in improving ovarian reserve but this translates into an improved reproductive outcome in a population, previously limited to oocyte donation. Trial registration number REF/2022/01/051033

Study question Do levels of Oxidative stress (OS) biomarkers differ in the follicular fluid of women with endometriosis compared to tubal factor infertility? Summary answer 8-Isoprostane is a promising biomarker in diagnosing oxidative stress in women with endometriosis opening a window of opportunity for improving outcomes. What is known already A delicate balance between oxidants and antioxidants is required for oocyte development and maturation. Any perturbation in the oocyte microenvironment which is the follicular fluid results in hampered oocyte quality and further affects the embryo development. As endometriosis causes destruction of the oocyte milieu, various OS biomarkers have been explored with inconclusive results. The present study aimed to compare these biomarkers in follicular fluid of women with endometriosis and tubal factor infertility undergoing in-vitro fertilization and correlate with ART outcomes. Study design, size, duration In a cross sectional study conducted at the assisted conception unit at a tertiary care hospital from 2017-2018 women undergoing their first IVF cycles for endometriosis and tubal factor infertility were recruited in the study. The study included a total of 107 patients with 50 in the endometriosis group (Group1) and 57 in the tubal group (Group 2). Participants/materials, setting, methods Follicular fluid from women undergoing IVF in both the groups was collected and evaluated for Oxidative stress biomarkers including Reactive Oxygen species (ROS), Total Antioxidant Capacity (TAC), 8-Isoprostane (8-IP) levels. ROS levels were detected by chemiluminescence and TAC and 8-IP using enzyme immunoassays. Main results and the role of chance Levels of ROS {(39.6 cpm vs 54.8 cpm) (p = 0.2)}were comparable in both the groups. Levels of 8-IP were significantly higher in group 1 as compared to the group 2 {(85.2  pg/ul vs 39.2  pg/ul) (p = 0.01)}. TAC levels were higher in endometriosis group compared to the tubal group {(5.4 mM of Trolox vs 4.1 mM of Trolox) (p = 0.005)} which could explain comparable ongoing clinical pregnancy rate in both groups (24.0% vs 24.5%, p = 0.34). There was no correlation of OS biomarkers with ART outcomes in tubal factor. Follicular fluid in Group 1 revealed a negative correlation of ROS with cleavage rate (r = - 0.42, p = 0.002) and positive correlation of TAC with fertilization rate (r = 0.44, p = 0.001) and Good grade embryos (r = 0.4, p = 0.001). Limitations, reasons for caution The main limitation is the inability to assess the dietary intake and blood oxidative stress biomarkers in both the groups. Wider implications of the findings OS biomarkers especially 8-IP could be useful in prognosticating IVF outcomes in women with endometriosis with a potential role of addition of antioxidants in culture media during IVF in these women. Trial registration number Not Applicable

Study question What is the ploidy concordance rate between spent culture media (SCM) and trophectoderm biopsy (TE) using Next Generation Sequencing (NGS) and correlation with clinical outcome? Summary answer DNA could be isolated from SCM with successful NGS library sequencing. Ploidy and per-chromosome concordance rate of TE biopsy vs SCM was 68.38% and 85.13%. What is known already Many challenges are associated with TE biopsy, and the possibility of NiPGT-A using cell free DNA (cfDNA) from SCM is very intriguing, as it will be simpler, safer and cheaper alternative to the gold standard TE biopsy. The bottlenecks associated with TE biopsy like additional equipment and trained embryologist, can be substantially reduced. The disparities in reported results in various studies comparing SCM vs TE, could be attributed to culture media contamination (maternal, paternal or environment), different laboratory workflow methodologies used for embryo culture, different whole genome amplification methods, library sequencing methods and different algorithms for analysis. Study design, size, duration Prospective cohort study was conducted in tertiary care centre from August 2021-June 2022. Ethical Approval was taken from Institute Ethics Committee. Couples with female age ≥ 35 years, one or more implantation failures, male factor infertility requiring ICSI, opting for elective single euploid blastocyst transfer, were included in study. Forty four blastocysts were obtained from 14 patients who underwent IVF/ICSI cycles, gave consent for TE biopsy and SCM collection for PGT-A and NiPGT-A respectively. Participants/materials, setting, methods Individualised ovarian stimulation was carried out with GnRH antagonist protocol. ICSI was done for fertilization. Embryos were cultured in sequential medium. On day 3, after thorough washing embryo was cultured separately in 10μl micro-droplets. SCM was collected on day-5, before biopsy. DNA extraction, amplification and library preparation from TE biopsy and SCM was done using Veriseq PGS kit (Illumina) and sequencing was done on Illumina MiSeq system. Euploid embryo transfer was done in FET cycle Main results and the role of chance Out of 44 blastocysts, 31 were day 5 and 13 were day 6. WGA-DNA from TE biopsy and SCM was 100% successful. Average concentration of amplified DNA obtained from TE biopsy and SCM was 34.88 ± 9.56 ng/µl, 32.3 ± 6.84 ng/µl respectively. Quality control parameters for library preparation and sequencing for samples were as per recommended standards. CNV visualization and analysis was done using BlueFuse Multi-Software (Illumina). Ploidy concordance rate could be analysed in 26 embryos, which was 68.38%, per chromosome concordance was 85.13% and sex-chromosome concordance was 73.0%. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of NiPGT-A was 66.6%, 60%, 87.5%, 30% and 65.38% respectively. On comparing day 5 and day 6 blastocysts, day 6 had better concordance rate 72.7% vs 60%, p > 0.05. On comparing good (>BB) and poor morphology (<BB)embryos, <BB had better concordance rate 83.33% vs 50%, p = 0.16. Per chromosome concordance rate was higher for <BB embryos, 90.5% vs 80.5%,p= 0.001. Full maternal cell contamination was suspected when TE was aneuploid/mosaic, XY and SCM was euploid, XX (n = 4/14), assessed only in XY embryos. Single euploid blastocyst transfer had 66.6%clinical pregnancy rate. One resulted in healthy live-birth, two on-going pregnancies, 30weeks and 12weeks period of gestation. Limitations, reasons for caution Before considering NiPGT-A in routine clinical practice, each embryology lab needs standardization, to implement necessary modifications in routine embryology protocol. Labs should ensure that embryo viability is not affected and should try to minimize chances of maternal or external contamination in SCM to reliably predict concordance rates with high confidence. Wider implications of the findings NiPGT-A is useful technique to assess ploidy but presently, NiPGT-A in combination with PGT-A, can help in prioritizing embryos according to their implantation potential. Further studies are required to find out whether NiPGT-A may serve as an alternative to PGT-A and implemented alone in routine clinical practice in future. Trial registration number CTRI/2021/04/033121