Treatment with Calcium Ionophore Improves The Results in Patients with Previous Unsuccessful Attempts at The Fertilization: A Cohort Study.

BACKGROUND: The objective of this study is to evaluate artificial oocyte activation (AOA) with calcium ionophore (CaI) in a subsequent attempt at fertilisation in patients after extremely low or failed fertilisation. We assessed improvements in fertilisation, implantation and pregnancy rates as well as cancellation rates in these patients. Finally, was evaluated the result testing in addition to delivery rate and obstetric outcomes in children born after AOA. MATERIALS AND METHODS: This was a retrospective observational study conducted in an IVF laboratory of an IVI clinic (IVIRMA Valencia, Spain). One group (509 mature oocytes from 66 patients) received a first intracytoplasmic sperm injection (ICSI) without AOA, which resulted in either a failed fertilisation or very low values (<30%). This group was compared with a second group (616 mature oocytes from the same 66 patients) that used AOA. Outcome was compared by McNemar's test and the dependent t tests. RESULTS: AOA plus CaI resulted in enhanced fertilisation (51 vs. 13.1%), ongoing pregnancy (47 vs. 21.7%), and implantation (31.1 vs. 13.1%) rates, and less chances for cancelling the cycle (22.7 vs. 69.3%). There were no observed adverse effects in obstetric and perinatal outcomes after the use of AOA. CONCLUSION: Our findings support the use of AOA for a given population of patients where fertilisation was affected during previous attempts. After AOA, we observed a significant increase in reproductive success due to the increased number of embryos available for embryo selection and, therefore, enhanced chances for success. The use of this artificial technique is comforting after checking non-existence of detrimental effects on the offspring.

Single-cell RNA sequencing of oocytes from ovarian endometriosis patients reveals a differential transcriptomic profile associated with lower quality.

STUDY QUESTION: Do oocytes from women with ovarian endometriosis (OE) have a different transcriptomic profile than those from healthy women? SUMMARY ANSWER: Oocytes from endometriosis patients, independently of whether they came from the affected ovary, exhibited a differential transcriptomic profile compared to oocytes from healthy egg donors. WHAT IS KNOWN ALREADY: Studies of endometriosis have sought to determine whether OE affects oocyte quality. While many reports indicate that oocytes recovered from endometriotic ovaries may be affected by the disease, other studies have found no significant differences among oocyte/embryo quality and fertilization, implantation and pregnancy rates in women with endometriosis. STUDY DESIGN, SIZE, DURATION: This prospective study compared metaphase II (MII) oocytes (n = 16) from endometriosis patients (n = 7) to oocytes (n = 16) from healthy egg donors (n = 5) by single-cell RNA sequencing (scRNA-seq). Participants were recruited between December 2016 and February 2018 at IVI-RMA Valencia and Vigo clinics. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human MII oocytes were collected from healthy egg donors and OE patients aged 18-34 years, with a body mass index of <30 and >6 pre-antral follicles. RNA was extracted, cDNA was generated and libraries were constructed and sequenced. scRNA-seq data libraries were processed and statistically analysed. Selected genes were validated by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Our scRNA-seq results revealed an effect of endometriosis on global transcriptome behaviour in oocytes from endometriotic ovaries. The highest number of differentially expressed genes (DEGs) was found when oocytes from women with OE were compared to oocytes from healthy donors [520 DEGs (394 upregulated and 126 downregulated)], independently of whether oocytes came from an affected or unaffected ovary. Among the top 20 significant DEGs in this comparison, most were upregulated, including APOE, DUSP1, G0S2, H2AFZ, ID4, MGST1 and WEE1. PXK was the only downregulated gene. Subsequently, functional analysis showed 31 enriched functions deregulated in endometriosis patients (Benjamini P < 0.1), being 16 significant enriched functions considering Benjamini P < 0.05, which involved in biological processes and molecular functions, such as steroid metabolism, response to oxidative stress and cell growth regulation. In addition, our functional analysis showed enrichment for mitochondria, which are an important cellular component in oocyte development. Other functions important in embryo development, such as angiogenesis and methylation, were also significantly enriched. LARGE SCALE DATA: All raw sequencing data are submitted in Gene Expression Omnibus (GEO) under accession number (PRJNA514416). LIMITATIONS, REASONS FOR CAUTION: This study was restricted only to OE and thereby other anatomical entities, such as peritoneal and deep infiltrating endometriosis, were not considered. This is a descriptive study with a limited number of samples reflecting the difficulty to recruit human oocytes, especially from women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that OE exhibits a global transcriptomic effect on oocytes of patients in OE, independently if they come from an affected or unaffected ovary and alters key biological processes and molecular functions related to steroid metabolism, response to oxidative stress and cell growth regulation, which reduce oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by IVI Foundation, the Spanish Ministry of Economy and Competitiveness through the Miguel Servet programme (CPII018/00002 to F.D.), the Sara Borrell Program (CD15/00057 to H.F.) and the VALi+d Programe (Generalitat Valenciana); ACIF/2016/444 to A.C.). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: None.

The use of morphokinetic as a predictor of implantation.

In recent years the increased efforts intended for improving future outcomes in the laboratory have focused mostly on the search of additional markers of embryo quality to add up present embryo selection criteria. Time-lapse system involves an alternative tool in assisted reproduction techniques, being able to improve the embryo selection from a dynamic and interactive approach while standard embryo assessment implies a subjective and static morphology evaluation and consequently reducing the information gained for embryo selection, time-lapse technology adds several morphokinetic parameters, providing additional input for embryo evaluation. This further information represents a challenge for a potential improvement in implantation rates and reproductive outcomes. This article focuses on the different time-lapse systems burgeoning on the market and the use of morphokinetics as a predictor of embryo implantation.

Combination of metabolism measurement and a time-lapse system provides an embryo selection method based on oxygen uptake and chronology of cytokinesis timing.

OBJECTIVE: To evaluate correlations between oxygen consumption (OC) measurements before and after embryo cytokinesis, observing OC during embryo cleavages and combining that information with morphokinetics to relate to implantation potential. DESIGN: Prospective cohort study. SETTING: University-affiliated private IVF unit. PATIENT(S): A total of 1,150 injected oocytes in 86 first oocyte donation cycles with embryo transfer on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): We analyzed the embryo OC and combined this data with the cytokinesis event, exact timing (in hours) of blastomeric cleavages, with the use of an incubator equipped with time-lapse videography, gathering a total of 7,630 measurements during the cytokinesis (active phase) and consecutive measurements after this division (passive phase), correlating this data with embryo outcome. RESULT(S): OC was found to increase during embryo cleavage, showing high levels during first division with a strong correlation with implantation success. Moreover, those embryos with slow or fast development gave rise to lower OC levels, whereas higher levels were associated with optimal embryo division ranges linked to higher implantation potential. CONCLUSION(S): A detailed analysis of OC by time-lapse observations enhances the value that these measurements represented as markers of embryo quality, especially during the cytokinesis events produced during preimplantation development.

A time to look back: analysis of morphokinetic characteristics of human embryo development.

OBJECTIVE: To describe the times associated with the morphological changes that occur in the embryo during preimplantation development based on the largest sample size described with time lapse. DESIGN: Cohort study. SETTING: University-affiliated private center. PATIENT(S): A total of 9,530 embryos from 1,806 intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using a time-lapse system, embryo images were acquired for at least 68 hours, in some cases reaching 120-130 hours. Embryo cleavage time points up to 8-cell-stage (t2-t8) as well as morulae (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle (cc) and synchrony (s) of the second and third cell cycles were defined. Finally, four subgroups of embryos were considered: the "regular divisions" group excluded embryos with a direct cleavage from 1 to 3 or 2 to 5 cells, and the "viable 8-cell," the "viable blastocyst," and "implanted embryos" groups included only embryos viable to the 8-cell stage, blastocyst stage, or transferred and successfully implanted, respectively. RESULT(S): Averages of times in the general population were: t2 = 27.9 hours, t3 = 38.2 hours, t4 = 40.7 hours, t5 = 51.0 hours, t6 = 54.1 hours, t7 = 56.7 hours, t8 = 59.1 hours, tM = 86.6 hours, tB = 104.1 hours, cc2 = 10.3 hours, cc3 = 12.8 hours, s2 = 2.7 hours, and s3 = 9.9 hours. Comparison between groups showed significant differences between regular divisions and viable 8 cells for t2, t3, t5, cc2, cc3, s2, and s3; between 8 cells and blastocyst for t5, t8, tM, cc3, and s2; and between blastocyst and implanted embryos for t8, tM, tB, and s2. Differences in timing related to morphology of cleavage- and blastocyst-stage embryos were detected. CONCLUSION(S): A time-lapse monitoring system applied to embryology allows accuracy and objectivity when defining the basis of embryo development within a clinic. The sample size is the largest ever described that provides consistent information about the normal distribution of embryo developmental timings.

Time-dependent O2 consumption patterns determined optimal time ranges for selecting viable human embryos.

OBJECTIVE: To evaluate correlations between metabolic activity and implantation potential of transferred embryos in a study based on oxygen (O(2)) consumption (OC) measurements, because O(2) uptake is directly related to the capacity of an embryo to produce energy via adenosine triphosphate. DESIGN: Retrospective cohort study. SETTING: Infertility institute. PATIENT(S): Five hundred seventy-five injected oocytes in 56 first oocyte donation cycles with embryo transfer on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We analyzed embryo destination viability and implantation depending on the embryo OC rate obtained from 47,741 measurements (up to 85 measurements per embryo, 2-3 measurements per hour). OC patterns were analyzed in relation to the time elapsed from sperm microinjection, to the final destination of the embryos (transferred, frozen, or discarded), to ongoing pregnancy, and by successful implantation. RESULT(S): OC was found to decrease during embryonic development. OC patterns from 52 hours onward showed the strongest correlation with implantation success. Regarding embryo destination, the same patterns were observed. CONCLUSION(S): OC from individual embryos revealed significant differences, mainly close to the time of transfer, when OC pattern was associated with successful implantation. Therefore, measuring the OC pattern of human embryos culture up to 72 hours could be used to select the embryo with best developmental potential.

The use of morphokinetics as a predictor of embryo implantation.

BACKGROUND: Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. METHODS: Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. RESULTS: A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. CONCLUSIONS: The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.

Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

OBJECTIVE: To check the effectiveness of intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA) in a globozoospermic patient. DESIGN: Case report. SETTING: Instituto Valenciano de Infertilidad, Valencia, Spain. PATIENT(S): A patient with globozoospermia. INTERVENTION(S): ICSI was administered in 14 oocytes. ICSI combined with AOA, in which a small amount of calcium was injected followed by calcium ionophore exposure, was done in 9 oocytes. MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality was assessed in both groups. RESULT(S): Chemical activation increased fertilization rate (55.6% vs. 35.7%) and the number of embryos with less multinucleation on day 2 (0 vs. 60%). Two embryos generated from AOA were transferred into the uterus (on day 3), resulting in a pregnancy and a healthy newborn. CONCLUSION(S): The AOA with calcium ionophore treatment improved fertilization rate and quality of the embryos, and was found to be an effective method for AOA in this patient with a low fertilization rate after previous ICSI treatment.