Oocyte cryopreservation review: outcomes of medical oocyte cryopreservation and planned oocyte cryopreservation

Background The utilization of oocyte cryopreservation (OC) has become popularized with increasing numbers of reproductive-aged patients desiring to maintain fertility for future family building. OC was initially used for fertility preservation in postmenarchal patients prior to gonadotoxic therapies; however, it is now available to patients to circumvent age-related infertility and other diagnoses associated with early loss of ovarian reserve. The primary aim of this paper is to provide a narrative review of the most recent and robust data on the utilization and outcomes of OC in both patient populations. Summary OC results in similar oocyte yield in patients facing gonadotoxic therapies and patients undergoing planned OC. Available data are insufficient to predict the live birth rates or the number of oocytes needed to result in live birth. However, oocyte yield and live birth rates are best among patients < 37.5 years old or with anti-mullerian hormone levels > 1.995 ng/dL, at the time of oocyte retrieval. There is a high ‘no use’ rate (58.9%) in patients using planned OC with 62.5% returning to use frozen oocytes with a spouse. The utilization rate in medical OC patients is < 10%. There is currently no data on the effects of BMI, smoking, or ethnicity on planned OC outcomes. Conclusion It is too early to draw any final conclusions on outcomes of OC in medical OC and planned OC; however, preliminary data supports that utilization of OC in both groups result in preservation of fertility and subsequent live births in patients who return to use their cryopreserved eggs. Higher oocyte yield, with fewer ovarian stimulation cycles, and higher live birth rates are seen in patients who seek OC at younger ages, reinforcing the importance of age on fertility preservation. More studies are needed in medical OC and planned OC to help guide counseling and decision-making in patients seeking these services.

Improving Implantation Rate in 2nd ICSI Cycle through Ovarian Stimulation with FSH and LH in GNRH Antagonist Regimen

Objective To investigate whether patients with a previous recombinant follicle stimulating hormone (rFSH)-stimulated cycle would have improved outcomes with rFSH + recombinant luteinizing hormone (rLH) stimulation in the following cycle. Methods For the present retrospective case-control study, 228 cycles performed in 114 patients undergoing intracytoplasmic sperm injection (ICSI) between 2015 and 2018 in an in vitro fertilization (IVF) center were evaluated. Controlled ovarian stimulation (COS) was achieved with rFSH (Gonal-f, Serono, Geneva, Switzerland) in the first ICSI cycle (rFSH group), and with rFSH and rLH (Pergoveris, Merck Serono S.p.A, Bari, Italy) in the second cycle (rFSH + rLH group). The ICSI outcomes were compared among the groups. Results Higher estradiol levels, oocyte yield, day-3 high-quality embryos rate and implantation rate, and a lower miscarriage rate were observed in the rFSH + rLH group compared with the rFSH group. In patients < 35 years old, the implantation rate was higher in the rFSH + rLH group compared with the rFSH group. In patients ≥ 35 years old, higher estradiol levels, oocyte yield, day-3 high-quality embryos rate, and implantation rate were observed in the rFSH + rLH group. In patients with ≤ 4 retrieved oocytes, oocyte yield, mature oocytes rate, normal cleavage speed, implantation rate, and miscarriage rate were improved in the rFSH + rLH group. In patients with ≥ 5 retrieved oocytes, higher estradiol levels, oocyte yield, and implantation rate were observed in the rFSH + rLH group. Conclusion Ovarian stimulation with luteinizing hormone (LH) supplementation results in higher implantation rates, independent of maternal age and response to COS when compared with previous cycles stimulated with rFSH only. Improvements were also observed for ICSI outcomes and miscarriage after stratification by age and retrieved oocytes.

Correlation between estradiol levels on day of HCG trigger and the number of mature follicles, number of oocytes retrieved, and the number of mature oocytes (M2) after oocyte aspiration in ICSI cycles

Background Estradiol is an important marker of ovarian response to ovarian stimulation in ART cycles. The study tries to find the correlation of serum estradiol on the day of HCG trigger to the number of follicles, the number of oocytes retrieved, and the number of mature oocytes, and also, to correlate estradiol per follicle and estradiol per oocyte on the day of HCG, to the number of oocytes retrieved, and to the number of mature oocytes. It is a cross sectional study using retrospective data. Results The data of 232 patients were analyzed. Our study showed a positive correlation between estradiol levels and the number of follicles (NF) (r = 0.592, p < 0.001), number of retrieved oocytes (NRO) (r = 0.576, p < 0.001), and number of mature oocytes (NMO) (r = 0.554, p < 0.001). E/follicle ratio did not have a significant correlation with NRO and NMO. E/Oocyte ratio had a strong negative correlation with NMO (r = −0.280, p < 0.001) Conclusions Serum estradiol had a positive correlation with NF, NRO, and NMO. But E/O had a strong negative correlation with NMO. These results indicate that estradiol levels can be used as an important clinical tool in the prediction of oocyte and mature oocyte yield in ART cycles. Reproductive outcome in ART cycles is largely dependent on the number of oocytes and mature oocyte yield. Estradiol levels on the day of HCG appear to strongly correlate with the outcome of ART cycles.

P–464 What is the optimal ovarian stimulation (OS) protocol for women who undergo planned oocyte cryopreservation (POC)?

Study question When repeated cycles of OS for planned oocyte cryopreservation using a standard GnRH antagonist protocol are required, can OS protocol modifications improve oocyte yield? Summary answer Compared to repeating a standard GnRH antagonist protocol, switching to a long GnRH agonist protocol for POC results in a higher number of cryopreserved oocytes. What is known already The total number of cryopreserved oocytes is a key parameter of POC programs because of its association with livebirth. A substantial proportion of women embarking on POC will undergo repeated cycles of OS to reach their desired target number of vitrified oocytes. According to recent guidelines, the GnRH antagonist protocol with GnRH agonist triggering is considered the first choice protocol for POC, because of its safety profile and convenience. However, in women with normal ovarian reserve, the long GnRH agonist protocol results in a higher number of oocytes retrieved. Evidence regarding the optimal protocol for POC is limited. Study design, size, duration This is a single-centre, retrospective cohort study including 283 women who had a first cycle for POC using a standard GnRH antagonist protocol and who requested a second OS cycle to increase their total number of vitrified oocytes for later use. The choice of protocol for the second cycle was left at the discretion of the reproductive medicine specialist. All OS cycles took place between January 2009 and December 2019 in a tertiary referral hospital. Participants/materials, setting, methods After ovarian reserve testing, the first cycle OS was performed using rFSH or HPhMG in a GnRH antagonist protocol. For the second cycle, a GnRH antagonist protocol with or without antagonist pretreatment, or a long GnRH agonist protocol was prescribed. The primary outcome was the number of mature oocytes (MII) vitrified per cycle. Cycle characteristics were compared. Data were assessed by generalized estimating equation (GEE) regression analysis adjusting for covariates. Main results and the role of chance In total, 226 (79.9%) women had a GnRH antagonist protocol and 57 (20.1%) had a long GnRH agonist protocol in their second OS cycle for POC. Overall, mean age was 36.6±2.4 years. The median (CI) number of mature oocytes vitrified after the second OS cycle was significantly higher than that after the first cycle [8 (5–11) vs. 7 (4–10), p &lt; 0.001]. According to GEE multivariate regression, adjusting for relevant confounders, switching from a GnRH antagonist protocol in the first cycle to a long GnRH agonist protocol in the second cycle was the only significant predictor of the number of vitrified oocytes after the subsequent cycle (coefficient 1.59, CI 0.29–2.89, p-value = 0.017). Age, AFC, initial dose and type of gonadotropins did not predict the number of vitrified oocytes. None of the women developed moderate or severe OHSS. Similarly, of 174 women who underwent their first OS cycle with a standard GnRH antagonist protocol, 133 women (76.4%) had the same protocol for their second cycle and 41 women (23.6%) an additional three-day course of GnRH antagonist pretreatment. According to GEE multivariate regression, this protocol modification did not result in more mature oocytes available for vitrification (coefficient –0.25, CI –1.86–1.36, p-value = 0.76). Limitations, reasons for caution These data should be interpreted with caution because of the retrospective design and limited sample. Although more oocytes were obtained with a long GnRH agonist protocol we have no data on livebirth in women returning to use their oocytes to support the choice for a specific OS protocol for POC. Wider implications of the findings: Although oocyte yield in the context of POC is an important parameter that may be modulated by the choice of OS protocol, the ultimate outcome measure of a successful POC program is livebirth after oocyte vitrification. Future research of oocyte parameters reflecting oocyte quality is paramount. Trial registration number Not applicable

P–093 The use of donor sperm improves post-ICSI live birth rates in advanced maternal age women

Study question Can the use of donor sperm improve post-ICSI live birth rate in advanced maternal age (AMA) patients? Summary answer The use of donor sperm increases post-ICSI live birth rate while substantially reducing abortion occurrence in AMA patients. What is known already Oocyte DNA repair capacity decreases with maternal age, when sperm DNA integrity is particularly important to avoid the transfer of gene truncations and de novo mutations to the zygote. Optimal DNA repair activity in the zygote requires paternal inheritance of 8-oxoguanine DNA glycosylase (OGG1), a rate-limiting enzyme in the base excision repair pathway. However, the involvement of paternal aging and sperm quality in the severe drop in fertility observed in AMA patients has not been addressed. While strategies to mitigate the impact of AMA on fertility have exclusively targeted oocyte quality, the sperm contribution in this scenario remains somehow neglected. Study design, size, duration Retrospective, multicentric, international study including 755 first ICSI cycles with patients’ own oocytes achieving a fresh ET between 2015 and 2019, 337 of which using normozoospermic partner semen and 418 using donor sperm. The association of sperm origin (partner vs. donor) with live birth was assessed by univariate/multivariate analysis in non-AMA (&lt;37 years, n = 278) and AMA (≥37 years, n = 477) patients. ICSI outcomes were compared between partner and donor sperm in non-AMA and AMA patients. Participants/materials, setting, methods The study was conducted in 3 fertility clinics including 755 Caucasian patients aged 24 to 42 years. Univariate/multivariate analyses were performed to test the association of sperm origin with live birth; infertility factor, maternal age, oocyte yield and number of embryos transferred were included in the model as confounding variables. In addition, ICSI outcomes were compared between donor and partner sperm groups with the Chi-square (percentages) or with the Wilcoxon sum rank (continuous variables) tests. Main results and the role of chance The multivariate analysis revealed that the use of donor sperm was positively and independently associated with live birth occurrence in AMA [1.82 OR (1.08–3.07) 95% IC; p = 0.024], but not in non-AMA patients [1.53 (0.94–2.51); p = 0.090]. Maternal age [0.75 (0.64–0.87); p &lt; 0.001], number of MII oocytes recovered [1.14 (1.05–1.23); p = 0.001] and number of embryos transferred [1.90 (1.27–2.86); p = 0.002] were also independently associated with live birth in AMA patients. Live birth and delivery rates were 70–75% higher, while miscarriage rate was less than half in donor sperm compared to partner sperm AMA cycles (LBR: 25.4% vs. 14.5%, p = 0.003; DR: 22.5% vs. 13.5%, p = 0.008; MR: 18.0% vs. 39.5%; p = 0.009). Implantation (17.4% vs. 13.5%; p = 0.075) and clinical pregnancy rates (27.5% vs. 22.3%; p = 0.121) did not significantly differ between sperm donation and partner sperm AMA cycles. Male age was substantially lower (23.6 ± 5.2 vs. 41.4 ± 5.0; p &lt; 0.0001) and oocyte yield was higher (5.1 ± 3.1 vs. 4.3 ± 2.6; p &lt; 0.0001) in sperm donation compared to partner sperm AMA cycles, while maternal age did not vary (39.8 ± 1.6 vs. 39.6 ± 1.7; p = 0.348). Limitations, reasons for caution This study is limited by its retrospective nature and by differences in patients’ profiles between sperm donation and homologous cycles, although this variation has been controlled for in the statistical analysis. Wider implications of the findings: The findings suggest that donor sperm can improve live birth rates by drastically reducing miscarriage occurrence in AMA patients. Therefore, the present results may influence AMA treatment decisions and, above all, contribute for AMA patients to achieve a healthy birth. Trial registration number Not applicable

P–453 Fertility preservation in endometriosis: Impact of the ovarian endometriosis and it’s surgical treatment on oocyte yield

Study question Does a previous history of surgery for ovarian endometriosis (OMA) has an impact on controlled ovarian stimulation (COS) response in case of fertility preservation (FP) for endometriosis? Summary answer After COS, a prior history for OMA surgery was associated with poorer ovarian responsiveness compared to non-previously operated women. What is known already Endometriosis is a chronic disorder that affects 10% of woman, which can be responsible for infertility. The presence of OMA and/or it’s excision could induce a reduction of the ovarian reserve (ROR), and for some women, an increased risk of premature ovarian failure. Therefore, FP with oocyte/embryo vitrification can be proposed for OMA-affected women, considering the relationship between endometriosis, infertility and ROR. Although a complete surgery excision of endometriosis lesions may be appropriate for some patients to relieve them from pain, the more efficient time to preserve fertility is still unknown in the management of women presenting OMA lesions. Study design, size, duration We conducted an observational multicentric study from April 2015 to December 2019, in two tertiary care university hospitals. Women presenting OMA or having a previous history of surgery for OMA that had performed a FP with COS for oocytes/embryo vitrification during the study period were included. Diagnosis of endometriosis was based on published imaging criteria using transvaginal sonography and magnetic resonance imaging or histologically proven in women who had past surgery. Participants/materials, setting, methods A total of 165 women were allocated to two groups, according to the presence of a previous history of surgery for endometrioma(s). Main outcome measure was the total number of oocytes retrieved. Main results and the role of chance Fifty-one (30,9%) women were included in the group ‘previous history of surgery’ and 115 (69,1%) in the group ‘no history of surgery’. Mean age was 31,6±4,4 years and was not significantly different between groups (p = 0.09). However, women in ‘No previous surgery’ group had higher AMH levels than women in ‘previous surgery’ group (2.27±1.70ng/ml versus 1.56±1.89ng/ml; p &lt; 0,001). In the group ‘previous history of surgery’, 21(41.2%) women had a recurrence of OMA(s) and 31 (60.8%) had at least one deep infiltrating endometriosis (DIE) lesion at FP. In the group ‘no history of surgery’, 92(80.7%) of the women had DIE. In addition, women in ‘No previous surgery group’ had larger OMA than women in ‘previous surgery’ group (mean diameter size: 5.56±4.34cm versus 3.25±2.16cm, respectively; p:0,03). The mean number of COS with oocyte-retrieval was significantly higher in the group ‘previous history of surgery’ (2.0±1.02 versus 1.65±0.82 in the group ‘no surgery’, p = 0.03), however, the total number of oocytes retrieved per women was significantly higher in women ‘history of surgery’, compared to women ‘no previous surgery’ (13.7±8.4 versus 10.3±7.5, p = 0.02). In addition, the cancellation rate per cycle was significantly lower in ‘No previous surgery’ group compared to the ‘previous surgery’ group (0.09±0.31 versus 0.28±0.53; p &lt; 0.001). Limitations, reasons for caution No data concerning the thawing of oocytes/embryo are available for now. Wider implications of the findings: FP is an essential component to integrate in ovarian endometriosis-management and should be proposed before surgery to optimize oocyte yield. Trial registration number Not applicable

P–806 Neogametogenesis via oocyte replication

Study question Can full preimplantation embryo development be achieved from artificial oocytes created through nuclear transfer of a haploid pseudo-blastomere (HpB) into a recipient ooplast? Summary answer It is feasible to replicate the female genome and generate novel sibling oocytes that can yield full preimplantation embryo development, albeit at a reduced rate. What is known already A limitation of assisted reproduction is the number of available oocytes for embryo creation. It is feasible to utilize a somatic cell nucleus to construct novel oocytes through a process known as haploidization, in which a reverse meiosis occurs after SCNT. Similarly, producing haploid parthenogenetic constructs can generate HpBs, useful for genetic testing at the pre-fertilization level or for reproduction. It is feasible to use a HpB as a nuclear donor since it has already completed homologue segregation. Study design, size, duration This is prospective translational animal model study. Over 6 months, 556 oocytes were manipulated for the experimental group, and 158 control oocytes were employed. B6D2F1 HpBs were used to establish the procedure and acquire expertise. FVB HpBs were subsequently introduced for genetic variance. Experimental and control embryos were cultured in a time-lapse incubator (up to 96h). Cleavage parameters were compared to control. Two-sample T-tests and one-way ANOVA with Bonferroni correction were employed for statistical analysis. Participants/materials, setting, methods A cohort of oocytes was harvested from B6D2F1 or FVB superovulated mice and artificially activated by 8% ethanol. At the 8-cell stage, HpBs were exposed to nocodazole. Another cohort of B6D2F1 oocytes was enucleated for recipient ooplasts. HpBs were individually transferred into the perivitelline space of the ooplasts alongside inactivated Sendai virus. After fusion, reconstructed oocytes with spindle development were fertilized by piezo-actuated ICSI using B6D2F1 spermatozoa. Unmanipulated and fertilized B6D2F1 oocytes served as control. Main results and the role of chance A total of 158 control oocytes underwent ICSI with a 67.7% survival rate; of these, 65.4% developed to the blastocyst stage. For artificial oocyte activation (AOA), up to 10 oocytes were activated for each experiment, yielding 8 HpBs per activated oocyte. For the experimental group, 556 oocytes underwent enucleation with a 96.4% survival rate. Nuclear transfer of HpBs resulted in a 93.2% survival rate, consistent for those derived from BDF and FVB. Reconstructed oocytes showed appropriate development of a novel pseudo-meoitic spindle at a rate of 63.7% for B6D2F1 HpBs and 75.5% for FVB HpBs, and ICSI yielded a 67.1% and 57.7% survival rate, respectively. The fertilization rate for the reconstructed oocytes was 64%. Control oocytes underwent ICSI with a 67.7% survival rate. When evaluating time-lapse parameters, reconstructed embryos created via blastomere nuclear transfer showed asynchrony compared to controls beginning as early as the stage of pronuclear fading. While the majority of reconstructed embryos arrested at the 4-cell stage, of those that progressed, 11.3% of those using BDF HpBs and 14.6% of those using FVB HpBs developed to the fully expanded blastocyst stage. This corresponds to a total of 23 reconstructed embryos that developed to the morula or blastocyst stage. Limitations, reasons for caution While we used single-well embryoscope culture for morphokinetic data collection, group culture is superior to single-embryo culture for mice. Thus, developmental rates may be underestimated by this protocol. Implantation and successful pregnancy are also needed to support the clinical utility of this method in generating gametes. Wider implications of the findings: For women with diminished ovarian reserve, oocyte yield and age-related aneuploidy are limitations to achieving genotyped offspring. Nuclear transfer of HpB can generate sibling oocytes while maintaining genetic information. This model represents a promising path for expanding oocyte yield, allowing genetic assessment of sibling oocytes, and enhancing chances of procreation. Trial registration number none

P–628 Predicting oocyte yield in patients with diminished ovarian reserve using basal antral follicle count

Study question Is basal antral follicle count (bAFC) taken on day 1 to 3 of stimulation a useful predictor of oocyte yield in that cycle, in women with diminished ovarian reserve (DOR)? Summary answer Basal AFC has moderate correlation with final oocyte yield. A median 75% of the antral follicle count is collected as oocytes. What is known already The current theory of folliculogenesis suggests that all follicles available for recruitment are visible on ultrasound in the ovary at the point when ovarian stimulation is applied. This implies a tight correlation between the AFC on day 1–3 of a stimulation cycle (bAFC) and the eventual number of follicles collected. We hypothesise that in women with diminished ovarian reserve who receive maximum stimulation basal AFC would be a useful predictor of final oocyte yield in that cycle. Study design, size, duration This was a prospective single centre, observational study in a tertiary referral hospital in London. 125 women with DOR underwent controlled ovarian stimulation between December 2018 and January 2021. Participants/materials, setting, methods All study participants were given an antagonist cycle with a starting stimulation dose of 450iu and remained on the same dose throughout their treatment. We assessed the correlation between bAFC taken on day 1–3 of the stimulation cycle and the total number of oocytes collected. Main results and the role of chance A total of 150 treatment cycles were included in the analysis. The median age was 37 (IQR 35 – 39). The median AMH was 6.0 (IQR 4.4 – 8.9) and the median FSH was 7.6 (IQR 5.7 – 9.4). The median bAFC at the start was 9 (IQR 6 – 11). The median total stimulation dose was 4050iu (IQR 4050 – 4500). The median oestradiol on day of trigger was 5906 (IQR 4166 – 7397) and median number of oocytes collected was 7 (IQR 5 – 9). There was a moderate correlation between bAFC and the number of oocytes collected (r = 0.549, p = 0.005). The median ratio of oocytes collected over the number of antral follicles observed at the start was 72.7% (IQR 58.3 – 100). Limitations, reasons for caution We have standardised approach to AFC determination and have previously shown that AFC inter and intra-observer variability in our unit is low. Nevertheless, our study involved multiple operators for AFC determination which may introduce variability. Further variability may have been introduced at egg collection by varying technique. Wider implications of the findings: Studies of antagonist protocol in good prognosis patients suggest poor correlation between basal AFC and oocyte yield. In contrast, our study shows that in a population of women with DOR basal AFC provides useful information which can be used to counsel women around the expected oocyte yield of their cycle. Trial registration number Not applicable