In vitro maturation of immature oocytes for fertility preservation in cancer patients compared to control patients with fertility problems in an in vitro fertilization program

Background The aim of this study was to determine whether in vitro maturation (IVM) of immature oocytes after controlled hormonal stimulation of the ovaries could be important in cancer patients to improve their chances of conception in the future. Patients and methods After ovarian stimulation in cancer patients, the number of oocytes and their quality and maturity were compared to control patients with fertility problems in the in vitro fertilization (IVF) program. In both groups of patients, immature oocytes at the developmental stage of germinal vesicle were matured in vitro and the proportion of oocytes that matured in vitro was compared between groups. In a subset of women with fertility problems, intracytoplasmic sperm injection (ICSI) was performed on IVM oocytes to assess their ability to be fertilized and develop into an embryo compared to vivo matured oocytes in the same cycles and consider the procedure in cancer patients. Results In patients with different cancers, the disease did not affect the number and quality of retrieved oocytes. In cancer patients, there was even a significantly lower proportion of immature oocytes than in patients with fertility problems (30.0% vs. 43.6%; P < 0.05). However, in patients with cancer, fewer oocytes per patient matured in vitro than in patients with fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83; P < 0.05). After ICSI, the proportions of fertilized oocytes and fertilized oocytes developing into an embryo did not differ between oocytes matured in vitro and in vivo in the same cycles. Conclusions Oocyte IVM is proving to be a reliable procedure for resolving immature oocytes after controlled ovarian stimulation in cancer patients.

In Vitro Maturation of Oocytes Retrieved from Ovarian Tissue: Outcomes from Current Approaches and Future Perspectives

In vitro maturation (IVM) of transvaginally aspirated immature oocytes is an effective and safe assisted reproductive treatment for predicted or high responder patients. Currently, immature oocytes are also being collected from the contralateral ovary during laparoscopy/laparotomy and even ex vivo from the excised ovary or the spent media during ovarian tissue preparation prior to ovarian cortex cryopreservation. The first live births from in vitro-matured ovarian tissue oocytes (OTO-IVM) were reported after monophasic OTO-IVM, showing the ability to achieve mature OTO-IVM oocytes. However, fertilisations rates and further embryological developmental capacity appeared impaired. The introduction of a biphasic IVM, also called capacitation (CAPA)-IVM, has been a significant improvement of the oocytes maturation protocol. However, evidence on OTO-IVM is still scarce and validation of the first results is of utmost importance to confirm reproducibility, including the follow-up of OTO-IVM children. Differences between IVM and OTO-IVM should be well understood to provide realistic expectations to patients.

Histidine buffered media maintains pH stabile during cooled transportation of human ovarian tissue

The aim of this study was to investigate whether pH is stable when transporting ovarian tissue in media buffered with either HEPES or histidine. Furthermore, if the choice of transport media impacts the in vitro maturation rate of oocytes collected in connection with ovarian tissue cryopreservation. Human ovaries (n = 34) collected for ovarian tissue cryopreservation were transported immersed in either 30 ml of HEPES buffered (follicle flushing media (Origio; Denmark)) or histidine buffered media (Custodiol®-HTK, Koehler-Chemie, Germany). Tissue was transported on ice for 4–5 h. At arrival, the ovary was weighed, and the pH of the media was measured at 0 °C. From 15 patients, immature oocytes were collected for in vitro maturation, oocytes that matured to metaphase II were evaluated. The pH measured in the HEPES buffered media (pH = 7.5 ± 0.13, n = 18) was significantly higher (p < 0.001) than the pH measured in the histidine buffered media (pH = 7.2 ± 0.05, n = 16). The standard deviation of pH measurements for the histidine buffered media was significantly lower than for the HEPES buffered media measurements (p < 0.0001). A total of 170 and 247 immature oocytes were collected and in vitro matured from ovaries transported in HEPES and histidine buffered media, respectively. The maturation rate of immature oocytes after IVM was similar in the two groups. The results show that pH in the histidine buffered media is closer to the physiological level and more stable than in HEPES buffered medium and support the use of histidine buffered media for cooled transportation of human ovaries.

New Insights in Canine Reproduction

Reproduction in canids has been characterized by having some peculiar aspects, such as the extended reproductive cycle and ovulation of immature oocytes [...]

P–271 Should intracytoplasmic sperm injection (ICSI) of delayed mature oocytes become a routine practice in the IVF Laboratory?

Study question Do delayed mature oocytes result in similar euploid blastocyst rates as their immediate mature sibling oocytes? Summary answer Once a blastocyst is obtained, delayed mature oocytes have similar euploid rates compared to immediate mature oocytes. What is known already Intracytoplasmic sperm injection (ICSI) of metaphase II oocytes few hours post oocyte retrieval is standard practice in IVF laboratories. Immature metaphase I (MI) and prophase I (GV) oocytes are usually discarded. Immature oocytes may mature overnight, after which ICSI can be performed. Studies demonstrated lower fertilization and blastulation rates for these delayed mature oocytes. However, live births have been reported from blastocysts transferred. The evidence available is not compelling, since most of the studies had either low sample size, no preimplantation genetic testing for aneuploidies (PGT-A), or the outcome was not compared to sibling MII oocytes at time of denudation. Study design, size, duration A single-center retrospective sibling oocyte study was performed between January 2019 and December 2020 at ART Fertility clinics Abu Dhabi, UAE. A total of 345 PGT-A cycles, with at least one delayed mature oocyte inseminated by ICSI, were included: 2506 immediate mature oocytes and 669 delayed mature oocytes. Participants/materials, setting, methods Following controlled ovarian stimulation, MII oocytes at the time of denudation were inseminated by ICSI/IVF (immediate mature). Immature oocytes (MI/GV) were cultured for 16–24 hours in fertilization medium and injected the next day if matured (delayed mature). Trophectoderm biopsy was performed on day 5/6/7 and samples were subjected to Next Generation Sequencing to screen the ploidy state of the blastocyst. Main results and the role of chance The 345 controlled ovarian stimulation cycles resulted in the insemination of 2506 MII oocytes on the day of oocyte retrieval (Day0) and 669 delayed mature oocytes on day 1. Normal fertilization rate was significantly higher in the immediate mature oocytes compared to delayed mature oocytes (68% vs 56%, p &lt; 0.0001). Similarly, the usable blastocyst rate was significantly higher in immediate mature oocytes (59% vs 19%, p &lt; 0.0001). On day 5 of development, a significantly higher-good quality blastocyst formation rate was obtained from immediate mature oocytes (65% vs 27%, p &lt; 0.0001). The rate of good quality blastocyst on the day of biopsy was significantly higher in the immediate mature oocytes group (76% vs 62%, p &lt; 0.015). Fisher’s Exact Test was performed to compare the euploid rate of blastocysts biopsied on day 5/6/7 originating from immediate mature oocytes or sibling delayed mature oocytes. The euploid potential of blastocyst biopsied showed no significant difference between the two groups (p = 0.388). Limitations, reasons for caution The timing of MI/GV oocytes transition to MII stage was not recorded since the incubation was done in a benchtop incubator. Furthermore, the same sperm sample was used to inseminate immediate and delayed mature oocytes, which might contribute to the compromised embryo development due to increased sperm DNA fragmentation. Wider implications of the findings: Insemination of delayed mature oocytes by ICSI, should be considered as a tool to increase patients’ chances of obtaining a euploid embryo. Especially in cases where low yield of euploid embryos is expected. Trial registration number Not applicable

P–274 Undetectable viral RNA in follicular fluid (FF), cumulus cells (CC) and endometrial tissue in SARS-CoV–2 positive patients

Study question Is there any indication for presence of viral RNA in FF, CC, immature oocytes or endometrial biopsy (EB) of SARS-CoV–2 patients undergoing ovarian stimulation? Summary answer Viral RNA is undetectable in FF, CC and EB with RT-PCR. However, S-protein expression on corona radiata cells suggests susceptibility to SARS-CoV–2 infection. What is known already The effects of a SARS-CoV–2 infection on the female reproductive system are still poorly understood. Theoretically, co-localisation of the angiotensin converting enzyme (ACE2) and transmembrane serine protease 2 (TMPRSS2) on human blastocysts implies susceptibility to viral infection, mediated by the coronavirus spike (S) protein. To date, SARS-CoV–2 RNA was undetectable in mature oocytes from COVID–19 patients, despite the expression of ACE2 and TMPRSS2. The presence of viral RNA in endometrial tissue, immature oocytes, CC or FF has not yet been investigated in samples from patients with positive nasopharyngeal SARS-CoV–2 test. Study design, size, duration This is a prospective, single-centre, observational study including ten patients with a positive nasopharyngeal swab for SARS-CoV–2, performed 48 hours before oocyte retrieval (OR), from September 2020 to January 2021. A patient was eligible if she preferred to continue treatment following adequate counselling of the unknown but presumably low risk for vertical transmission. Since a freeze-all strategy was applied, an EB was performed. Participants/materials, setting, methods During OR, all protective measures were taken. Pooled FF, CC and EB from each patient were tested for viral RNA presence with RealStar® SARS-CoV–2 RT-PCR-Kit1.0 (Altona-Diagnostics). Ct values &lt;40 were considered positive. EB was collected for pathological evaluation and cultured to obtain endometrial stromal cells (EnSC). Immature oocytes and EnSC were tested for S-protein expression by immunohistochemistry with anti-S antibody (MA5–35958, Thermo-Fisher Scientific) followed by Alexa Fluor™ 488-donkey-anti-mouse (Thermo-Fisher Scientific) and visualized with confocal microscopy. Main results and the role of chance SARS-CoV–2 RNA was undetectable in the pooled FF, CC and EB from all patients included in the study. Histological analysis of the EB showed no pathological modifications, including inflammatory reaction, as compared to biopsies collected from swab negative patients. After staining with anti-S antibody, cultured EnSC and immature oocytes tested negative for the S-protein. However, the binding of anti-S antibody was demonstrated on the corona radiata cells remaining on the zona pellucida after oocyte denudation for intra-cytoplasmatic sperm injection, indicating presence of SARS-CoV–2. In that case, the explanation for the undetectable viral RNA in CC could be that the viral RNA concentration remained under the detection limit of the currently used RT-PCR test. Limitations, reasons for caution This study was conducted in a small population (ten patients included) with different viral load, with mild or without symptoms of COVID–19. Another important limitation is the absence of validation of the RT-PCR protocol for the investigation of other types of samples than nasopharyngeal swabs. Wider implications of the findings: The absence of SARS-CoV–2 RNA in all samples analysed represents a step further in reassuring a safe ART program for COVID–19 patients. However, the presence of S-protein on corona radiata cells warrants further investigation, since the theoretical possibility to infect human oocytes and/or embryos cannot be ruled out. Trial registration number NCT04425317

P–192 Efficacy of postponement of intracytoplasmic sperm injection timing after spindle visualization for Metaphase I oocytes

Study question Does postponement of intracytoplasmic sperm injection (ICSI) timing after spindle visualization for Metaphase I (MI) oocytes improve developmental outcomes of embryos? Summary answer Postponement of ICSI timing after spindle visualization for MI oocytes improves blastocyst utility rates. What is known already Immature oocytes are generally considered poor developmental outcomes. Meanwhile, the timing of ICSI adjusted by using spindle visualization can improve clinically utilized embryos and live birth rates, but these outcomes remain inferior to those of mature oocytes. In in vitro maturation culture, nuclear maturation is thought to occur before the completion of cytoplasmic maturation, and in immature oocytes, synchronization of nuclear and cytoplasmic maturation may be insufficient for ICSI immediately after spindle visualization. Study design, size, duration Data for this retrospective cohort study were obtained 672 oocytes retrieved under mild stimulation cycles using letrozole, in patients aged younger than 39 years between April 2017 and October 2020.Written informed consent was obtained from all patients. This study was approved by the institutional review board. Participants/materials, setting, methods As a control group, 464 MetaphaseIIoocytes that underwent ICSI immediately after visualization of the spindle were used. In group A, 103 MI oocytes underwent ICSI immediately after the first polar body release and spindle visualization, and in group B, 105 oocytes underwent ICSI 2–3 hours after spindle visualization. The primary outcomes were fertilization rates, degeneration, cleavage, embryo blastocyst formation, and utility rates. Outcomes were compared among the three groups. Main results and the role of chance The baseline fertilization rates of each group (control, A, B) were 82.3% (382/464), 73.8% (76/103), and 83.8% (88/105), respectively. The rate was significantly lower in group A than in the control group (P &lt; 0.05), and also tended to be lower in group A than in group B, although the difference was not significant. There was no significant difference in abnormal fertilization rates, oocyte degeneration rates, cleavage rates, and blastocyst formation rates among the three groups. [control, A, B: abnormal fertilization rate: 4.3% (20/464), 8.7% (9/103), 4.8% (5/105); oocyte degeneration rates: 3.0% (14/464), 1.9% (2/103), 3.8% (4/105); cleavage rates: 95.6% (307/321), 93.8% (61/65), 98.7% (74/75); blastocyst formation rates: 58.6% (177/302), 51.7% (31/60), 55.4% (41/74), respectively]. The blastocyst utility rates of control group and group B were significantly higher than in group A [41.7% (126/302), 45.9% (34/74), 26.7% (16/60), respectively] (P &lt; 0.05). There were no significantly different outcomes between the control group and group B. Limitations, reasons for caution The optimal timing of ICSI for MI oocyte cannot be determined by the presence or absence of spindles. In addition, the postponement duration we set was based on reports which reported on final oocyte maturation, and further investigation is needed to establish the optimal ICSI timing for MI oocytes. Wider implications of the findings: In MI oocytes, postponement of ICSI timing after spindle visualization is essential for synchronization of the nucleus and cytoplasmic maturation. Trial registration number none

P–239 In vitro maturation of human oocytes: a systematic review and data analysis

Study question Based on published studies, how effective is in vitro maturation (IVM) in different patient groups, and how does the maturation rate correlate with culture conditions? Summary answer Clinical IVM is most effective when patients receive only hCG trigger prior to oocyte collection. Multiple additional parameters influencing the outcome were identified. What is known already Despite being used for more than fifty years, the overall efficacy of human IVM has not yet been determined, and results are often conflicting. Indeed, IVM is still perceived skeptically by many embryologists and doctors and not widely used in clinical practice. This review aims to collect all available data in the literature regarding the efficacy of IVM analyzing characteristics of patients, treatment, or laboratory conditions that may influence the MII-rate (MR). Study design, size, duration: A systematic search was performed in the PubMed database following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search was limited to studies in the English language published before October 2020 using the following keyword: “oocyte in vitro maturation». Participants/materials, setting, methods Inclusion criteria for studies were: reporting data obtained on immature human oocytes, which transitioned to the MII stage after IVM. The requirement was that the numbers of cultured and matured oocytes were reported. If available, additional data were collected including patients’ characteristics (for example PCOS), hormonal stimulation prior to the procedure (administration of some FSH or hCG trigger or both), oocyte freezing before or after IVM, type of culture medium and supplements, etc. Main results and the role of chance A total of 350 publications were selected from 6866 search results, 436 abstracts, and 422 full read articles. Selected studies cover 21153 patients and 157420 immature oocytes cultured. It has been demonstrated that oocytes collected in vivo from adult, non-PCOS patients, who received only hCG trigger prior to the procedure had a statistically higher MII rate (66%) than oocytes from patients who received no gonadotropins or some FSH, or a combination of some FSH and hCG trigger (59%, 60% and 58% respectively). The same was valid for PCOS patients: MR in the trigger only cohort (66%) was significantly different from other cohorts. MR for in vivo collected oocytes (61%) from adult non-PCOS patients was significantly different from ex vivo collected oocytes (33%). MI stage oocytes at the moment of collection matured with a statistically higher rate (N = 4322, 73%), than GV oocytes (N = 3328, 54%). When in vitro matured oocytes were vitrified, their average survival rate was 81% (data from 50 studies on 1701 oocytes). Additionally, immature oocytes survived vitrification with a 75% rate (data from 30 studies on 4457 oocytes). Overall, ICSI fertilization rate for IVM oocytes was 69% (N = 59914). A total of 747 babies born from IVM were reported. Limitations, reasons for caution Among selected publications only 2 were randomized controlled trials and therefore the main challenge of this review is striking differences in setups among included studies. However, despite not being a meta-analysis, this study calculated MR for the most frequent treatment modalities and additional individual factors, which might influence MR. Wider implications of the findings: This review provides data regarding IVM efficiency in different cohorts of patients, performed under different culture conditions. Additional laboratory parameters influencing MR have been identified. Based on this new data, target groups benefiting the most were identified, and prognosis regarding the success of their treatment with IVM might be estimated. Trial registration number n/a

P–709 Dual stimulation in-vitro-maturation (Duostim IVM) for overcoming oocyte maturation arrest, resulting in embryo transfer and livebirth

Study question Does luteal phase followed by follicular phase letrozole priming and dual oocyte retrieval for in-vitro maturation (IVM) overcome oocyte maturation arrest (OMA)? Summary answer Oocyte maturation, fertilization,embryo cryopreservation and livebirth can be achieved with letrozole priming IVM in rare cases of OMA. What is known already OMA is an intractable problem resulting in only immature oocytes being collected and to date no succesful treatment exists. Attempts to mature oocytes collected in stimulated IVF cycles with OMA have so far failed. Cases with OMA can be due to intrinsic oocyte defects, intrafollicular factors or resistance to stimulation. Study design, size, duration Six women with OMA in ≥ 2 prior stimulated IVF cycles were treated between March 2019 and December 2020. Participants/materials, setting, methods Participants had total of 18 (range 2 - 6) prior IVF cycles yielding only 166 immature oocytes. Letrozole 5mg was given days 15–18 of ovulatory cycle; SC decapeptyl 0.1mg trigger given at follicles 12 mm, 38 hours&lt;OPU. After menstruation, letrozole 5mg days 3–7; SChCG 250ug when follicles=12 mm 38 hours&lt;OPU. After in-vitro-maturation oocytes reaching MII were fertilized. Embryos from luteal collection were frozen and fresh embryo transfer was attempted after follicular phase collection. Main results and the role of chance Six women underwent DuoStim IVM, median (quartiles) 3.5 (0 - 9) GV and 0.5 (0 - 2) MI oocytes were collected from luteal phase OC and 0 (0 - 0) GV and 2(0 – 4.5) MI oocytes were collected from follicular phase OC. They had a total of 166 immature oocytes collected in prior IVF cycles. There were no MII oocytes at the time of collection in any cycles.0 (0 – 3.5) oocytes matured from luteal phase OC and 1 (0 – 4) from follicular phase OC. 0 (0 – 1.5) embryos were available from luteal phase and 0 (0 - 2) from follicular phase OC.Two subjects (29 and 33 years old) underwent fresh DET and the 29 year old with 2 previous failed IVF cycles achieved a livebirth (50% per ET and 16.7% per started cycle). None of the women who did not have an embryo for fresh transfer from the follicular phase collection had an embryo from the luteal phase collection. The same 29 year old has 2 luteal phase and 2 more follicular phase embryos vitrified. Limitations, reasons for caution OMA is a rare condition with a variety of etiologies. Different etiologies can require different managements. Wider implications of the findings: It may be possible to overcome OMA with letrozole IVM in rare cases. This case is the first recorded live birth. The value of dual stimulation overcoming OMA remains uncertain. Trial registration number This study is approved by the local ethical commitee of Medicana Samsun International Hospital by a Grant number of 02/05.02.2020: registration is not required due to retrospective status