Frozen-thawed Oocytes Research Articles

Use of tamoxifene-controlled ovarian hyperstimulation for fertility preservation before breast cancer treatment: A prospective cohort study with a 5-year follow-up

PurposeFertility issues are of great concern for young women undergoing treatment for breast cancer (BC). Fertility preservation (FP) protocols using controlled ovarian stimulation (COS) with letrozole have been widely used with overall good results. However, letrozole cannot be used in every country in this context. This study aimed to assess the efficacy of tamoxifen for COS in women with early BC undergoing FP. MethodsThis multicentric prospective study included patients aged 18–40, diagnosed with stage I, II and III invasive BC, undergoing tamoxifen-COS before adjuvant or neoadjuvant chemotherapy (NAC). The primary endpoint was the efficacy of tamoxifen-COS protocol evaluated by the number of oocytes collected and vitrified. Secondary endpoints included the time interval before chemotherapy, breast cancer (BC) recurrence rates, and reproductive outcomes. ResultsNinety-five patients were included between 2014 and 2017, aged 31.5 ± 4 years on average. 37.9 % received NAC and 62.1 % received adjuvant chemotherapy. FP procedure was successful in 89.5 % of the cycles. The mean number of collected and vitrified oocytes was 12.8 ± 7.9 and 9.8 ± 6.2, respectively. The mean duration of COS was 10.4 ± 1.9 days. Median time before chemotherapy initiation was 3.6 weeks (IQR 3.1; 4.1) for women receiving NAC. Five-year relapse-free and overall survival rates were in-line with those expected in this population. Twenty-one women had spontaneous full-term pregnancies, while 5 underwent IVF cycles with frozen-thawed oocytes, without pregnancy. ConclusionTamoxifen-COS protocols appear to be feasible before adjuvant or NAC treatment in young BC patients and efficient in terms of oocyte yield.

Preimplantation genetic testing for aneuploidy is associated with reduced live birth rates in fresh but not frozen donor oocyte in vitro fertilization cycles: an analysis of 18,562 donor cycles reported to Society for Assisted Reproductive Technology Clinic Outcome Reporting System

ObjectiveTo evaluate the impact of preimplantation genetic testing for aneuploidy (PGT-A) on first transfer live birth rate (LBR) and cumulative LBR (CLBR) in donor oocyte IVF cycles. DesignRetrospective cohort study of the SART CORS database. Subjects11,348 fresh and 7,214 frozen-thawed donor oocyte IVF cycles were analyzed. ExposureThe first reported donor stimulation cycle per patient between January 1, 2014 and December 31, 2015, and all linked embryo transfer cycles between January 1, 2014 and December 31, 2016, were included in the study. Main Outcome MeasuresLBR was compared for patients using fresh and frozen-thawed donor oocytes, with or without PGT-A. Logistic regression models were adjusted for age, body mass index, gravidity, infertility etiology, and prior IVF cycles. ResultsAmong patients who had blastocysts available for transfer or PGT-A, use of PGT-A was associated with a decreased first transfer LBR (46.9 vs 53.2%, p <0.001) and CLBR (58.4 vs 66.6%, p <0.001) in fresh oocyte donor cycles compared with no PGT-A. LBR in frozen-thawed oocyte donor cycles with PGT-A were nominally higher than those without PGTA (48.3% vs. 40.5%) but were not statistically significant in multivariable logistic regression models (p=0.14). Early pregnancy loss was not significantly different with and without PGT-A. Multiple gestation, preterm birth, and low birthweight infants were all reduced with addition of PGT-A in fresh donor oocyte cycles, though these outcomes were not significantly different when comparing single embryo transfers in fresh oocyte cycles and also not significantly different among frozen-thawed donor oocyte cycles. ConclusionPGT-A in fresh oocyte donor cycles was associated with decreased LBR and CLBR, while effects on frozen-thawed oocyte donor cycles were clinically negligible. Obstetrical benefits associated with PGT-A in fresh donor cycles appear linked to increased single embryo transfer.

P-221 The efficiency of a PGT-A program is negatively impacted by the utilization of frozen-thawed oocytes in patients of all ages

Abstract Study question Are there any differences in the use of fresh or frozen-thawed oocytes in a PGT-A program? Summary answer While euploidy rates are comparable, the efficiency of a PGT program is adversely impacted when utilizing frozen-thawed oocytes. What is known already Oocyte vitrification has become one of the key assisted reproduction procedures. Some studies have reported rates of embryonic development, clinical pregnancy, and embryo implantation comparable between fresh and cryopreserved oocytes in egg donor patients. Vitrified oocyte accumulation before the blastocyst biopsy is a commonly used strategy in PGT programs to enhance the chances of obtaining a euploid blastocyst with the patient’s own oocytes. Further research is needed to assess the impact of frozen-thawed oocytes on aneuploidy and the efficiency of PGT programs. Study design, size, duration We conducted a retrospective comparison of 777 PGT-A cycles, using either fresh or frozen-thawed oocytes, regarding embryo development (fertilization rates, blastocyst formation, and rates of good-quality blastocysts), PGT outcomes (euploidy and mosaicism rates), and the overall efficiency of the PGT cycle, measured as the number of euploid blastocyst per available mature oocytes, between April 2018 and November 2023. The study groups were stratified based on the age of the patients (≤35 and &amp;gt;35 years old). Participants/materials, setting, methods PGT-A cycles were categorized into two groups: patients who utilized fresh oocytes (n = 641) and those who utilized frozen-thawed oocytes (n = 136). A total of 6,370 fresh oocytes and surviving oocytes from the initially vitrified ones (972 out of 1,153) were fertilized and cultured to the blastocyst stage, at which point biopsies were performed. The biopsied cells were subjected to NGS screening, and statistical data analysis was conducted using the Chi-square test, with significance set at P &amp;lt; 0.05. Main results and the role of chance In the group of patients aged ≤35 years old, significant differences were observed when comparing PGT cycles using fresh or frozen-thawed oocytes in terms of the fertilization rate (78.4% vs. 72.0%), blastocyst formation rate (47.4% vs. 37.7%), and the efficiency of the PGT cycle (24.0% vs. 16.0%), corresponding to a 33.3% reduction in efficiency. No differences were observed between fresh and frozen-thawed oocytes regarding euploidy rate (64.6% vs. 68.4%), mosaicism rate (4.9% vs. 5.1%), and the percentage of good-quality embryos (60.0% vs. 56.1%). Similarly, in patients aged &amp;gt;35 years old, fertilization (81.5% vs. 73.5%), blastocyst formation (40.5% vs. 33.4%), and the efficiency of the PGT cycle (11.7% vs. 7.2%), which correspond to a 38.5% reduction in efficiency, were improved when using fresh oocytes. No differences were observed regarding euploidy (35.6% vs. 35.3%) or mosaicism rate (3.8% vs. 5.3%), and the percentage of good-quality embryos (55.2% vs. 54.7%). The oocyte survival rate was 84.3%. Limitations, reasons for caution Our study is limited by its sample size and retrospective design, as well as the inclusion of a non-selected and general infertile population, which could amplify the impact of confounding variables. Wider implications of the findings We found that the utilization of frozen-thawed oocytes reduces the PGT program efficiency by decreasing fertilization and blastocyst formation rates. This suggests that the oocyte accumulation strategy should be approached with caution. However, vitrification did not lead to an increase in the rates of aneuploidy, mosaicism, or poor-quality blastocysts. Trial registration number NOT APPLICABLE

O-247 Validation of an AI (Artificial intelligence) model assessing oocyte quality in a single donor program with frozen-thawed oocytes for blastocyst prediction

Abstract Study question Can an AI model predict blastocyst development in a specific subset population of frozen-thawed oocytes in a single donor program? Summary answer An AI oocyte quality score is predictive of blastocyst formation of frozen-thawed oocytes using post-ICSI images. What is known already The ability to score oocytes based on quality is of paramount importance. A methodology to categorize oocyte quality continues to elude IVF professionals. CHLOE OQ™ is an AI oocyte-quality (OQ) score designed to analyse a single oocyte Timelapse image and provide an OQ score. Responsible use of AI needs the validation of models across different clinical scenarios. The increasing use of frozen-thawed oocytes in IVF has necessitated to objectively score the developmental potential of this oocytes. The objective of this study was to determine if the morphological characteristics of frozen-thawed oocyte images are predictive of blastocyst formation. Study design, size, duration Retrospective cohort study using time-lapse videos of 1318 post-ICSI frozen-thawed donated oocytes between January 2021 and August 2023, with known blastulation outcomes from a single private clinic. Participants/materials, setting, methods Oocytes were evaluated using OQ Score and placed into categories according to their OQ score. Group A: &amp;gt;0.6, Group B: 0.3-0.59, Group C: 0.001-0.29 and Group D: 0. The correlation between OQ score, embryo quality (EQ) score and blastocyst development was measured with binary logistic regression (AUC) and a 2-sample t-test. Chi-square was performed to determine if there were significant differences in blastocyst development rates across these distinct oocyte quality groups. Main results and the role of chance Oocytes that underwent blastulation exhibited a significantly higher OQ Score (7.0 ± 2.3) compared to those that did not blastulate (5.3 ± 3.0, p &amp;lt; 0.001). The predictive capacity for blastocyst formation was observed for OQ score (AUC= 0.67) and EQ Score (AUC= 0.93), with a baseline blastulation of 66% (n = 1318, p &amp;lt; 0.001). Of interest, as early as Day 3 a Blastocyst prediction score was highly predictive of blastulation (AUC= 0.92, p &amp;lt; 0.001). When assessing a subset of transferred oocytes, OQ Score had a higher predictive ability (AUC=0.71, n = 163, p &amp;lt; 0.001).Oocyte quality scores within OQ score groups showed a direct association with blastocyst formation rate: [Group A 74% (656/878), Group B 56% (130/231), Group C 43% (70/164), Group D 13% (3/23), p &amp;lt; 0.05]. The highest-scoring group demonstrated a five-fold increase in blastulation rate compared to the lowest-scoring group (p &amp;lt; 0.001). Furthermore, the highest-scoring group resulted in the highest proportion of good-quality embryos compared to the lowest-scoring group [78% (456/582) vs 0.19% (1/582), p &amp;lt; 0.001]. Limitations, reasons for caution Retrospective assessment of a single clinic. Results reflect the use of frozen-thawed donated oocytes. Therefore, the proportion of data within the four oocyte quality groups is not equally distributed and is biased towards high quality oocytes. This may impact the predictive ability within the smaller subgroups. Wider implications of the findings Developing an objective methodology to categorize oocyte quality post-retrieval is crucial. It is important to validate AI models in different clinical scenarios, for example in oocyte donor programs using frozen-thawed oocytes. AI is a tool that can be used to predict blastocyst development rates within a frozen donor oocyte program. Trial registration number not applicable

P-379 Fertility outcomes several years after fertility preservation for endometriosis

Abstract Study question To study the fertility outcomes of women who tried to conceive at least 2 years after fertility preservation (FP) for endometriosis. Summary answer To advise patients with endometriosis on the benefit of FP, particulary on the utilization rates of frozen oocytes and the actual chances of pregnancy What is known already Endometriosis is found in 25-40% of infertile patients. Due to the multiple and recurrent nature of lesions and the significant incidence of repeated ovarian surgery, endometriosis increase the risk of damaging the ovarian follicular stockpile. Since the revision of the French bioethics law (2014), women with endometriosis can be offered FP techniques. However, there is currently little data available in this population, limiting the possibility to establish formal recommendations Study design, size, duration A retrospective observational, bicentric cohort study conducted at Antoine Beclère and Jean Verdier University Hospitals.Inclusion criteria were: i) patients with confirmed endometriosis using imaging (pelvic MRI or ultrasound scan); ii) controlled ovarian hyperstimulation for FP between from July 1st 2013, and December 31st, 2019 (n = 200). The endpoint date was February 15, 2023, with data collected retrospectively. Participants/materials, setting, methods Cumulative incidences of pregnancy and live birth were calculated. Time to conception, time to utilization of oocytes, and predictive factors for pregnancy and live birth were assessed using logistic regression Cox models. Main results and the role of chance Among the 184 patients with available data after FP for endometriosis, 76 attempted to conceive (41%). The mean number of oocytes vitrified per patient were 14.7 ± 7.6 [8-20]. Patients attempting conception at least 2 years post-FP were more likely to have a partner at the first consultation (70.7% vs. 25.7%, p &amp;lt; 0.0001). Overall, 48 patients (43%) achieved a pregnancy, predominantly through spontaneous conception (50%), frozen-thawed oocytes (37.5%), or a new IVF attempt using fresh eggs (12.5%). Among those attempting conception, 31 (39%) reported a live birth. Cumulative chances of pregnancy at 4 years post-FP were estimated at 52% CI 95% [41% - 65%]. Utilization rates for frozen oocyteswere 7% CI 95% [4% - 12%] and 18% CI 95% [13% - 26%], respectively, 2 and 5 years post-freezing. Nulliparity was the only factor significantly associated with an increased probability of oocyte utilization (RR = 1.43 [1.0 - 2.06]). Limitations, reasons for caution The result should be interpreted with caution due to the small sample size of our study. Data were collected retrospectively and some information, concerning obstetrical and neonatal information where missing. Furthermore, the duration of the follow up time was too short to evaluate long-term fertility. Wider implications of the findings Despite endometriosis affecting fertility, our study revealed that half of pregnancies occurred spontaneously, irrespective of the endometriosis phenotype. These findings provide valuable insights into advising women on the utilization rates of frozen oocytes (7% to 18%) and the relatively high chances of natural conception after FP for endometriosis. Trial registration number NA

P-359 Ten years’ experience using cryopreserved oocytes from In Vitro Maturation (IVM) or Controlled Ovarian Hyperstimulation (COH) cycles for fertility preservation in oncologic indications

Abstract Study question Is IVM a relevant option for fertility preservation in cancer patients, considering biological/clinical outcomes post-thawing, compared to COH? Summary answer This study shows that despite a significantly altered embryo morphology within the IVM group, the clinical outcomes remain comparable to those of COH. What is known already Fertility Preservation (FP) techniques are proposed to patients with cancer to provide the possibility of childbearing using their own gametes. Currently, oocyte vitrification after COH is the standard option. IVM serves as an alternative when COH is contraindicated, urgent cancer therapy is required, or in conjunction with ovarian cortex cryopreservation. Limited live births have been reported from frozen-thawed oocytes in IVM cycles of cancer survivors. Scarce data are available for comparing biological/clinical outcomes of frozen-thawed oocytes from IVM versus COH cycles in cancer survivors. Study design, size, duration This bicentric retrospective cohort study aimed to analyze the outcomes of all oocyte warming cycles in 73 cancer survivors having undergone oocyte vitrification for FP after COH and/or IVM. All of them had oocyte retrieval before the administration of gonadotoxic treatment and returned after being cured, with their oncologist agreement, for assisted reproduction treatments, between November 2012 and January 2024. Participants/materials, setting, methods Seventy-seven warming attempts followed by ICSI respectively from 37 COH and 40 IVM cycles were analyzed. Four patients benefited from an IVM and COH cycle. Survival, fertilization, top and good-quality embryos, defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation with &amp;lt; 20% of cytoplasmic fragments, implantation, biochemical (hCG&amp;gt;100 UI/mL), clinical (intrauterine sac with fetal heartbeat) and live birth rates were compared using appropriate statistical tests. Main results and the role of chance The mean age and antral follicle count at the time of FP was similar in both groups. The indications for FP were breast cancer (n = 61), hematologic malignancies (n = 7), ovarian cancer (n = 3), other (n = 2). The number of cryopreserved oocytes tended to be higher in the COH group, when compared to the IVM group (8.5±7.3 vs 5.9±4.04; p = 0.16). The mean oocyte maturation rate after IVM was 68.6±23.1%. Oocyte survival, and fertilization rates were similar in COH and IVM groups, respectively (76.4±27.0% vs 76.1±22.0%; p = 0.76) and (67.1±34.3% vs 65.3±25.4%; p = 0.36). We reported a significantly better embryo quality at day-2 in the COH group when compared to the IVM group, with respectively top-quality embryos (44.0±34.2% vs 26.1±33.4%, p = 0,04) and good-quality embryos (62.2±38.0% vs 42.8±40.8%, p = 0.049), and also a significantly higher number of useful embryo in the COH group (2.7±2.6 vs 1.5±1.2, p = 0.03). Regarding clinical outcomes, the results were similar in COH and IVM groups, in terms of implantation rates (12.2±30.4% vs 15.5±35.6%; p = 0.86), biochemical (32.3% (10/31) vs 17.9% (6/28); p = 0.39), clinical pregnancies (25.8% (8/31) vs 17.9% (4/28); p = 0.54), live birth rates ( 25.8% (8/31) vs 14.3% (4/28); p = 0.34). Limitations, reasons for caution Statistical power to compare IVF outcomes after COH and IVM is limited by the few women who returned to use their frozen oocytes. The subjective nature of embryo morphology assessment. Wider implications of the findings This study is the largest evaluating frozen-warmed oocytes from IVM cycles in cancer patients, with 5 live births newly reported. A higher oocyte-yield may be necessary in IVM as the number of useful embryos is significantly lower. Novel approaches are being developed to improve maturation rates following IVM. Trial registration number Not applicable

P-248 Egg vitrification related to female age: survival and laboratory outcome of 1233 vitrified-thawed oocytes in non-medical autologous use

Abstract Study question Is the female age at retrieval a determinant factor for the viability and potential of the thawed eggs? Summary answer In patients over 40, thawed oocytes display more abnormal fertilization, develop less in extended culture and yield less top quality blastocyst. What is known already Oocyte cryopreservation is a well-established procedure especially since the advent of the vitrification technique. The successful outcomes achieved with vitrification encourage its application in fertility preservation (FP) for medical and non-medical reasons. Besides, the age-related decline in women’s fertility and the oocyte quality negatively affect the outcome of assisted reproduction. Some studies have reported that age at oocyte retrieval strongly influences reproductive prognosis after FP. However, the appropriate age for non-medical FP is still debated. Although oocyte freezing at a younger age is recommended, there should be a balance between a favourable outcome and the cost-effectiveness of the strategy. Study design, size, duration Retrospective study on 166 patients with frozen-thawed oocytes who underwent ICSI during 2021-2023 in a single hospital. Oocyte vitrification had different purposes: banking for poor responders, to limit the number of embryos, and to solve unexpected situations where partner’s semen sample was unavailable. Patients were divided into three groups according to their age at retrieval (Group A ≤ 37 n = 77, Group B 37-40 n = 44, Group C ≥ 40 n = 45). Post-warming survival, fertilization, and embryological data were studied. Participants/materials, setting, methods 1233 oocytes were thawed. The intact oocytes were microinjected 1-2 hours after warming. Embryos were cultured in Sage 1-StepTM to blastocyst stage, and rated on daily basis. Embryo transfer was performed on day 3 or day 5 and remaining blastocysts were frozen on Day 5 or Day 6. Post warming survival, lysis after ICSI, fertilization, embryo development, blastulation rate (BR) and top blastulation rate (TBR), and morphokinetic features were studied in the 3 groups. Main results and the role of chance 84.6% of the 1233 oocytes (vitrified in a high-security closed system) survived the warming process. 1043 eggs were injected and the post-ICSI lysis rate was 11.2%. No significant differences were seen in thawing survival rate in groups A, B and C (85.6% vs. 83.5% vs. 83.8%, p=0.635) nor in the post-ICSI lysis rate (13.1% vs. 7.4% vs. 11.2%, p=0.072). 2PN fertilization rate was similar between the groups (67.8% vs. 61.3% vs. 67.7%, p=0.178) as well as the total number of embryos (303 vs 146 vs 188 p=0.605). However, triploid fertilization rate (3PN) statistically grew with age (2.8% vs. 5.8% vs. 6.5%, p=0.034). Significant differences were also reported in the global abnormal fertilisation rate (1PN+3PN) (6.4% vs 10.3% vs. 13.3%, p= 0.005). BR and TBR were statistically lower after the age of 40 (BR: 61.5% vs. 62.7% vs 40% p&amp;lt;0.001, TBR: 43.3% vs. 47.9% vs. 24.3% p&amp;lt;0.001). Morphokinetic features in the 3 groups were studied (abnormal cleavage pattern, tSB and tB). Overall results were similar between group A and group B but oocyte potential decreases drastically from the age of 40. Limitations, reasons for caution The major limitation of the study is its retrospective nature and the relative small size of each group. As most of patients were transferred during 2023, the pregnancies have not delivered yet so the live birth rate is not mentioned. Wider implications of the findings Studies have reported the impact of age at oocyte retrieval in case of medical FP in cancer and endometriosis. Here we report a negative effect on outcome after the age of 40 in non-medical FP. Patients should be informed that FP is not a certification of a successful outcome. Trial registration number not applicable

Embryonic, genetic and clinical outcomes of fresh versus vitrified oocyte: A retrospective cohort study

Objective: To compare embryonic development, ploidy status and clinical outcomes between fresh and frozen-thawed oocytes. Methods: This retrospective cohort study evaluated 83 fertilization cycles including both fresh and frozen oocytes from 79 patients at the HP Fertility Center of Hai Phong International Hospital of Obstetrics and Pediatrics in Vietnam. The patient underwent several ovarian stimulation cycles to accumulate a certain number of oocytes that would be vitrified. In the last oocyte retrieval, all patient's oocytes including both frozen and fresh would be fertilized. The outcomes included the rates of oocyte survival, cleavage embryo, blastocyst, ploidy status, pregnancy, biochemical pregnancy and clinical pregnancy. Results: The oocyte survival rate after thawing was 96.5%. No statistically significant difference was found when comparing fresh and frozen oocytes regarding fertilization rate (78.1% vs. 75.5%, P=0.461), usable cleavage embryo rate (86.9% vs. 87.2%, P=0.916) but usable blastocyst rate was found higher statistically in the frozen oocyte group (44.4% vs. 54.0%, P=0.049). The percentages of euploid, aneuploid and mosaic embryos between the fresh group and the vitrified group had no significant differences (33.8% vs. 31.6%, P=0.682; 51.0% vs. 54.2%, P=0.569; 15.2% vs. 12.4%, P=0.787; respectively). The rates of pregnancy, biochemical pregnancy and clinical pregnancy had no statistical difference (68.8% vs. 64.8%, P=0.764; 12.5% vs. 3.6%, P=0.258; 37.5% vs. 46.4%, P=0.565). 17 Mature oocytes are the minimum to have at least one euploid embryo. Conclusions: Oocyte vitrification does not affect embryonic, genetic and clinical results. The number of mature oocytes should be considered for fertilization in some cases.

P-716 The impact of the SARS-CoV-2 pandemic on ART activity in Italy during 2020

Study questionEvaluate the impact of restrictive measures, implemented to contain the spread SARS-CoV-2, on ART activity in Italy during 2020.Summary answerDue to periods of suspension of certain medical procedures in 2020, ART activity has undergone a substantial reduction.What is known alreadySince the end of February 2020, the spread of SARS-CoV-2 has taken a dramatic toll, especially in Northern Italy. In order to ensure that the majority of National Health System resources were employed to control the epidemic, non-urgent medical procedures such as ART treatments, were suspended or postponed by a Government Decree issued on March 9, 2020. Only fertility preservation in cancer patients continued as these procedures could not be deferred. The first suspension of activity lasted about two months. Then, throughout 2020 many ART centers were forced to suspend their activity again because of flairs in SARS-CoV-2 infection rates.Study design, size, durationThe Italian Assisted Reproductive Technology Register (IARTR) retrospectively analyzed ART cycles applied in Italy in 2020 (67,928 ART cycles performed on 57,423 patients). The 2020 ART cycles were compared to those applied in 2019 (82,476 ART cycles performed on 67,633 patients). Data collection was organized yearly in two different time frames: the first regards the activity conducted during the year until pregnancy and the second regards pregnancy outcomes.Participants/materials, setting, methodsA total of 205 ART clinics have sent data to the IARTR in 2019 and/or 2020. Among those clinics 54.1% were private, 37.1% were public and 8.9% were private covered by NHS. Records were securely kept in the IARTR archive. The analysis has been performed utilizing SPSS 27.0Main results and the role of chanceIn 2020, the number of total ART cycles reported was 67,928 including 38,728 homologous fresh cycles (Fresh), 1,099 frozen/thawed oocytes (FO), 19,314 frozen embryo (FER) and 8,787 ART donation cycles. In 2019, 82,476 cycles were reported, including 50,324 Fresh, 1,361 FO, 21,796 FER and 8,995 gametes donation. In 2020, there have been 14,548 less ART cycles than in 2019, equivalent to a 17.6% of reduction in overall activity. However, the reduction was not homogeneous for all ART techniques, in particular there was a reduction of -23% in fresh cycles, of -11.4% in FER, of -19.3% in FO and of -2.3% for gametes donation. This overall reduction led to 3,325 fewer pregnancies. The reduction of treatment cycles observed in 2020 brings to 2,119 fewer deliveries (-18.0%) and to 2,539 fewer live born babies (-19.8%) compared to 2019. The percentage reduction of births is not homogeneous for the different ART techniques, ranging from -0.3% in donation cycles to -36.9% in fresh cycles. Lastly, during 2020 a reduction in multiple births (-2.1%) has been observed.Limitations, reasons for cautionSince the national ART Register collect only summary data more deep analysis could not be performed. For some ART techniques the reduction may have been greater (i.e. donation cycles) if considering the growing trends of previous years.Wider implications of the findingsCovid-19 pandemic has had a strong impact on Italian Health System. ART procedures suffered a reduction of availability. Since Italy has the lowest birth rate in Europe and ART plays an important role answering to the need of infertile couples, Government should support access to these treatments with dedicated actions.Trial registration numbernot applicable

Membrane-permeable trehalose improves the freezing ability and developmental competence of in-vitro matured feline oocytes

Oocytes are highly sensitive to cryopreservation, which frequently results in an irreversible loss of developmental competence. We examined the effect of membrane-permeable trehalose on the freezing ability of feline oocytes matured in vitro. In Experiment 1, intracellular trehalose (trehalose hexaacetate; Tre-(OAc)6) was synthesized from trehalose precursor and subjected to spectroscopic characterization. The membrane permeability of the Tre-(OAc)6 was investigated by incubating oocytes with different concentrations of Tre-(OAc)6 (3, 15, and 30 mM). Optimum concentration and the toxicity of Tre-(OAc)6 were assessed in Experiment 2. The effects of Tre-(OAc)6 on freezing ability in terms of apoptotic gene expression and developmental competence of in-vitro matured oocytes were examined in Experiments 3 and 4, respectively. The Tre-(OAc)6 permeated into the ooplasm of cat oocytes in a dose- and time-dependent manner. The highest concentration of intracellular trehalose was detected when the oocytes were incubated for 24 h with 30 mM Tre-(OAc)6. For the toxicity test, incubation of oocytes with 3 mM Tre-(OAc)6 for 24 h did not affect maturation rate and embryo development. However, high doses of Tre-(OAc)6 (15 and 30 mM) significantly reduced maturation and fertilization rates (p < 0.05). In addition, frozen-thawed oocytes treated with 3 mM Tre-(OAc)6 significantly upregulated anti-apoptotic (BCL-2) gene expression compared with the control (0 mM) and other Tre-(OAc)6 concentrations (15 and 30 mM). Oocyte maturation in the presence of 3 mM Tre-(OAc)6 prior to cryopreservation significantly improved oocyte developmental competence in terms of cleavage and blastocyst rates when compared with the control group (p < 0.05). Our results lead us to infer that increasing the levels of intracellular trehalose by Tre-(OAc)6 during oocyte maturation improves the freezing ability of feline oocytes, albeit at specific concentrations.

Application of nanomaterial for maturation in vitro of cattle embryos

Aim. To evaluate the effectiveness of the use of nanomaterial in the environment for the further development of in vitro embryos derived from frozen-thawed oocytes in the system of conservation of genetic resources of animals at the cellular level. Methods. Biotechnological, cryobiological, morphological, cytogenetic, and statistical methods, as well as methods of statistical data processing were used in the research. Results. Oocyte-cumulus complexes (OCC) of cows were divided into four groups: three experimental, in which the maturation was performed in a medium containing 0.1, 0.01 and 0.001% UFS/sucrose and control - without the addition of nanobiomaterial. In vitro fertilization of pre-mature frozen-thawed ova of cows and subsequent maturation of embryos in the medium with the addition of UFS/sucrose (0.001%) showed an increase in the number of embryos by 16.7-22.1% compared with the addition of 0.1; 0.01% and 13.1% compared to the control group. It was found that the fragmentation rate of 2-cell cattle embryos decreased from 65.0 to 39.8% with a decrease in the concentration of UFS/sucrose from 0.1 to 0.001%. The most stable indicators of the fragmentation index from 78.4 to 50.0% were observed on the fourth day of embryo cultivation in experimental group B. Conclusions. Reducing the concentration of UFS/sucrose from 0.1 to 0.001% in the composition of the medium for in vitro maturation of cattle embryos leads to an increase of 16.7-22.1% in the number of embryos obtained.&#x0D; Keywords: oocyte-cumulus complex, cryopreservation, nanomaterial, in vitro maturation, embryo.

Assessment Viability of Frozen-Thawed Oocytes Using Non- Invasive Technique based on Buffalo Follicular Fluid

Assessment Viability of Frozen-Thawed Oocytes Using Non- Invasive Technique based on Buffalo Follicular Fluid

Do Strategies Favoring Frozen-thawed Embryo Transfer Have an Impact on Differences in IVF Success Rate, Multiple Pregnancy Rate, and Cost per Live Birth Between Fertility Clinics?

The aim of this retrospective cohort study is to establish whether strategies favoring frozen-thawed embryo transfer (FET) have an impact on differences in cumulative live birth rate (CLBR), weighted mean cost per live birth, and multiple pregnancy rate (MPR) between fertility clinics. Data were extracted from the Assisted Reproductive Technology (ART) Italian National Registry and refer to classical IVF or intracytoplasmic sperm injection (ICSI) cycles performed in 2018 in public funded Lombardy region fertility clinics. Propensity to FET cycles was expressed with the so-called freezing tendency index (FTI): [(No. of FETs + No. of transfers of embryos obtained from frozen-thawed oocytes) / (No. of fresh ETs + No. of canceled IVF cycles + Numerator)] × 100. Fertility clinics were divided according to their FTI: group A (FTI: 0-20%); group B (FTI: 20-40%); and group C (FTI: 40-60%). Groups A, B, and C included 6, 8, and 8 fertility clinics, respectively. The CLBR (95% CI) per started COH cycle was 13.1% (11.8-14.5%) in group A, 19.6% (18.6-20.7%) in group B, and 27.8% (26.8-28.9%) in group C (p < 0.0001). The weighted mean live birth cost was 32,770 ± 10,662 € in group A, 25,863 ± 11,510 € in group B, and 20,426 ± 3788 € in group C (p < 0.0001). The MPR (95% CI) was 15.4% (12.1-19.4%) in group A, 10.2% (8.7-12.1%) in group B (p = 0.0065), and 6.3% (5.2-7.6%) in group C (p = 0.0001). In conclusion, our results suggest that strategies favoring FET cycles are associated with an increased CLBR, a decreased weighted mean cost per live birth, and a decreased MPR.

Effect of Gamete Cryopreservation on miRNA Expression and Embryonic Development

Background: Fertility preservation is a meaningful option to retain fertility due to the medical or social needs. Sperm or oocytes preservation is the most extensive way for personal fertility preservation. Although the cryopreservation of sperm is well-used while the oocyte cryopreservation technology is improving, the effects and embryonic development potential of gamete cryopreservation need further investigation. Methods: The miRNAs expression of cryopreserved gamete was detected using miRNA sequencing in human and mouse, differentially expressed miRNAs (DEmiRs) were identified by comparing their expression in frozen-thawed and fresh gamete. Function of DEmiRs were detected by GO enrichment analysis, and common DEmiRs were validated by qRT-PCR. The embryonic developmental potential of cryopreserved sperm or oocytes were estimated in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases by retrospective cohort study. Results: 21 and 106 DEmiRs were detected in human and mouse frozen-thawed sperm compared with fresh group, respectively. miR-148b-3p and miR-30b-5p were validated that downregulation in both human and mouse frozen-thawed sperm, while miR-140-5p was upregulated, these 3 common DEmiRs were enriched in positive regulation of mitotic cell cycle, in utero embryonic development and others. Meanwhile, 285 DEmiRs were identified in mouse frozen-thawed oocytes, the number of DEmiRs was 2.61-fold than that in mouse sperm. The downregulation of miR-148b-3p and miR-30b-5p were also validated in mouse frozen-thawed oocytes. Clinic data of embryonic development which used cryopreserved sperm indicated the significant decrease of fertilization rete in both IVF and ICSI cases, as well as the decrease of blastocyst formation rate in ICSI cases, while the significant decrease of high qualified embryo rate was observed when used the cryopreserved oocytes compared with paired fresh oocytes which retrieved from the same female in ICSI cases, suggesting the potential negative effects of gamete cryopreservation on embryonic development. Conclusion: Cryopreservation could change the expression of miRNAs in both sperm and oocyte, and the altered miRNAs expression pattern further affected the fertilization and development of embryo. Funding Statement: This research was funded by National Key R&D Program of China (2017YFC1001300). The National Natural Science Foundation of China (81771651, 81873832). The Research Projects of Shanghai Science and Technology Commission (18411964300). Declaration of Interests: The authors declare there are no competing interests. Ethics Approval Statement: The animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Tongji University and the Ethics Committee of the Institute of Animal Science, Tongji University (TJAA06420101). Human sperm samples were donated for research from patients who had provided informed consent at Tongji hospital, with the approval of the Institutional Ethical Committee for Scientific Research (2019-062).

Oocyte Cryopreservation - Current Scenario and Future Perspectives: A Narrative Review.

Oocyte cryopreservation is a boon for women undergoing assisted reproductive technology. With the evolution in the technique of cryopreservation over the last three decades, there has been an exponential rise in the number of oocyte cryopreservation cycles for diverse indications. Apart from cancer patients, it has also been promoted as a mode of fertility insurance to overcome the age-related decline in fertility as well as post-surgical decline following endometriosis surgery. The objective of the review is to evaluate its clinical applications, ideal age at freezing, optimal oocyte number, freezing method of choice, efficacy, safety and recent advances. In the last decade, vitrification has surpassed slow freezing for oocyte cryopreservation. Although closed system of vitrification provides the aseptic environment, open vitrification is commonly followed in practice. Early to mid-thirties is a reasonable age group for planned oocyte cryopreservation, although it might be recommended at a younger age, in patients with diminished ovarian reserve. The patients should be motivated to preserve around 14–20 mature oocytes for successful live birth. Various studies have shown comparable fertilisation and pregnancy rates between Intracytoplasmic sperm injection with fresh and frozen-thawed oocytes. The available evidence has shown no increase in the incidence of congenital abnormalities in babies born through vitrified oocytes. In the future, image analysis using artificial intelligence, and spindle visualisation using poloscope may further enhance the outcome of oocyte cryopreservation.

Follicular extracellular vesicles enhance meiotic resumption of domestic cat vitrified oocytes

Extracellular vesicles (EVs) contain multiple factors that regulate cell and tissue function. However, understanding of their influence on gametes, including communication with the oocyte, remains limited. In the present study, we characterized the proteome of domestic cat (Felis catus) follicular fluid EVs (ffEV). To determine the influence of follicular fluid EVs on gamete cryosurvival and the ability to undergo in vitro maturation, cat oocytes were vitrified using the Cryotop method in the presence or absence of ffEV. Vitrified oocytes were thawed with or without ffEVs, assessed for survival, in vitro cultured for 26 hours and then evaluated for viability and meiotic status. Cat ffEVs had an average size of 129.3 ± 61.7 nm (mean ± SD) and characteristic doughnut shaped circular vesicles in transmission electron microscopy. Proteomic analyses of the ffEVs identified a total of 674 protein groups out of 1,974 proteins, which were classified as being involved in regulation of oxidative phosphorylation, extracellular matrix formation, oocyte meiosis, cholesterol metabolism, glycolysis/gluconeogenesis, and MAPK, PI3K-AKT, HIPPO and calcium signaling pathways. Furthermore, several chaperone proteins associated with the responses to osmotic and thermal stresses were also identified. There were no differences in the oocyte survival among fresh and vitrified oocyte; however, the addition of ffEVs to vitrification and/or thawing media enhanced the ability of frozen-thawed oocytes to resume meiosis. In summary, this study is the first to characterize protein content of cat ffEVs and their potential roles in sustaining meiotic competence of cryopreserved oocytes.

Bioinspired l-Proline Oligomers for the Cryopreservation of Oocytes via Controlling Ice Growth.

Various types of cells are routinely cryopreserved in modern regenerative and cell-based medicines. For instance, the oocyte is one of the most demanding cells to be cryopreserved in genetic engineering and human-assisted reproductive technology (ART). However, the usage of cryopreserved oocytes in ART clinics is still limited mainly because of the unstable survival rate. This is due to the fact that oocytes are more prone to be damaged by ice crystals in comparison to other cells, as oocytes are larger in size and surface area. Meanwhile, oocytes contain more water, and thus, ice crystals are easier to form inside the cells. Currently, to avoid injury by the formed ice crystals, cryopreservation (CP) of oocytes has to use large amounts of small molecules as cryoprotectants such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG), which can permeate into the cell and prevent ice formation inside. However, these molecules are chemically and epigenetically toxic to cells. Therefore, great efforts have been focused on reducing the amount of DMSO and EG used for oocyte CP. In nature, the antifreeze (glyco)proteins (AFGPs) locate extracellularly with the ability to protect living organisms from freezing damage via controlling ice growth. Inspired by this, biocompatible and nontoxic L-proline oligomers (L-Pron), which have the same polyproline II helix structure as that of AFGPs, are first employed for the CP of oocytes. The experimental results reveal that L-Pro8 has a profound activity in inhibiting ice growth as that of AFGP8. Also, by the addition of 50 mM L-Pro8, the amount of DMSO and EG can be greatly reduced by ca. 1.8 M for oocyte CP; moreover, the survival rate of the cryopreserved oocytes is increased up to 99.11%, and the coefficient of variance of the survival rate is decreased from 7.47 to 2.15%. These results mean that almost all oocytes can survive after CP with our method; importantly, the mitochondrial function as a critical criterion for the quality of the frozen-thawed oocytes is also improved. It is proposed that with the addition of L-Pro8, the extracellular ice growth is slowed down, which prevents the direct injuries of cells by large ice crystals and the accompanying osmotic pressure increase. As such, this work is not only significant for meeting the ever-increasing demand by the ART clinics but also gives guidance for designing materials in controlling ice growth during CP of other cells and tissues.

Enhancing the scope of in vitro maturation for fertility preservation: transvaginal retrieval of immature oocytes during endoscopic gynaecological procedures.

Could in vitro maturation (IVM) following transvaginal oocyte retrieval during gynaecological surgery (IVM-surgery) be an effective and safe strategy for fertility preservation? IVM-surgery on unstimulated ovaries is a novel option that can be considered for fertility preservation for women requiring gynaecological surgery, but more research is needed to identify appropriate patients who may benefit and to determine the cost-effectiveness of such an approach. IVM followed by oocyte/embryo cryopreservation has been useful as a safe reproductive strategy for some infertile women. This prospective cohort study comprised 158 consecutive women with polycystic ovary syndrome (PCOS) who underwent laparoscopy or hysteroscopy for other reasons and had concomitant transvaginal oocyte retrieval followed by IVM between 2014 and 2016. A total of 158 women with anovulatory PCOS who underwent IVM-surgery in our infertility centre were recruited for this study. Matured IVM oocytes obtained from these women were either freshly fertilized and subsequently frozen at the blastocyst stage (fresh oocyte group, n = 46) or the oocytes were frozen (frozen oocyte group, n = 112) for fertility preservation followed by later thawing for insemination and cleavage embryo transfer (ET) (n = 33). The following outcomes were then evaluated: embryological data, clinical pregnancy rate, live birth rate (LBR), neonatal outcomes, post-operative complications and post-operative ovarian function. Among all the women who underwent IVM-surgery, the clinical pregnancy rate and LBR per initiated IVM cycle were 9.5% (15/158) and 6.9% (11/158), respectively. Women (40.6%, 20/33) who underwent the procedure with frozen-thawed oocytes (oocyte survival rate, 83.0%) obtained a high quality of cleaved embryos. In the fresh oocyte group, the clinical pregnancy rate and LBR per ET cycle were 69.2 and 53.8%, respectively. In the frozen oocyte group, the clinical pregnancy rate and LBR per ET cycle were 28.6 and 19.1%, respectively. No adverse neonatal outcomes were recorded. IVM-surgery was not associated with post-operative complications, a longer hospital stay, or impaired ovarian function. Because of the small sample size and the low utilization rate and cost-effectiveness per retrieval, the present findings should be interpreted with caution, and further studies are needed for the long-term follow-up of live births. This strategy can also help patients with normal ovulation to obtain available oocytes and embryos for cryopreservation and subsequent use. This research was supported by the Joint Research Fund for Overseas Natural Science of China (No. 31429004), the National Key Research and Development Program of China (No. 2017YFC1002000, 2017YFC1001504, 2016YFC1000302), the Ministry of Science and Technology of China Grants (No. 2014CB943203), the Chinese Society of Reproductive Medicine Fund (No. 16020400656) and the National Natural Science Foundation of China (No. 81300456). All the authors have nothing to disclose in terms of conflicts of interest. chictr-ONC-17011861.

Oocyte Vitrification: A Comparative Analysis Between Fresh and Cryopreserved Oocytes in an Oocyte Donation Program

Background: Oocyte Cryopreservation has become an important part of infertility treatment for various reasons such as fertility preservation in women going for oncological treatment; in oocyte donation cycles; in eliminating several religious, ethical, and legal concerns of embryo freezing and in women who wish to delay childbirth. The newer ”vitrification” technique for freezing has further improved the success rates for actual conception than the earlier method of slow freezing. A successful oocyte freezing program can help in establishment of oocyte banks, which would help to provide compatible oocytes immediately, thus would eliminate the several problems of fresh donor cycles. Methods: In this retrospective observational study, total 60 oocyte donation cycles were included (38 were fresh and 22 were vitrified oocytes cycle, respectively). After a thorough screening, controlled ovarian hyperstimulation for donors was performed using flexible antagonist protocol. All mature oocytes were allocated into “vitrified oocytes” and “fresh oocytes” groups. Vitrification technique using Cryotop method was used for oocyte freezing. Both clinical and laboratory outcomes of vitrified and fresh oocytes in donor cycles were compared. Results: A total of 600 oocytes (226 “vitrified oocytes” and 374 fresh oocytes), were studied. After warming 218 oocytes survived resulting in survival rate of 96.4%. Fertilization rate and embryo formation rate was 86.2% and 93.6%, respectively. Results of frozen-thawed oocyte donor cycles were compared with fresh donation cycles. For fresh oocyte group, fertilization rate and embryo formation rate was 83.4% and 92.6%, respectively. On comparing clinical outcomes, clinical pregnancy rate was 60.5% in fresh group and 63.6% in vitrified group. Conclusions: Both clinical and laboratory results obtained in the study suggest that oocyte cryopreservation can be performed with reproducible success, thus vitrification technique can be provided as a useful tool for achieving highly successful outcomes in an oocyte donor program.

Failed sperm retrieval from severely oligospermic or non-obstructive azoospermic patients on oocyte retrieval day: Emergent oocyte cryopreservation is a feasible strategy.

PurposeUnexpected sperm retrieval failure on the day of oocyte retrieval is not common but frequently happened in patients with severe oligospermia or non-obstructive azoospermia(NOA). Oocyte cryopreservation is a common strategy after failed collection of sperm when concurrent ovarian stimulation is underwent. However, the use of oocyte vitrification in such male-infertility cases remains unclear.ObjectiveTo investigate the outcomes of emergent oocyte cryopreservation after failed sperm retrieval from severe oligospermic or non-obstructive azoospermic (NOA) patients on oocyte retrieval day.MethodsDesign: Retrospective cohort studySetting: Academic fertility center at Lee Women's Hospital, Taiwan, between March 2015 and August 2017.Patients: For 203 couples with NOA(n = 200) or severe oligospermia(n = 3), testicular spermatozoa (n = 67 cycles) or frozen donor sperm (n = 209 cycles) were injected into fresh or frozen-thawed oocytes via 276 intracytoplasmic sperm injection (ICSI) cycles.Main Outcome Measures: Clinical pregnancy and live-birth rates (LBRs).ResultsIn the 67 cycles involving the use of fresh testicular spermatozoa, no significant differences were observed between fresh and warmed oocytes with respect to the fertilization rates (69.2% vs. 74.1%; p = 0.27), number of Day-3 embryos (8.6±4.4 vs. 6.4±3.4; p = 0.08), number of good-quality Day-3 embryos (4.5±3.9vs. 4.7±3.0; p = 0.45), implantation rates (29.1% vs. 17.8%; p = 0.21), clinical pregnancy rates (36.4% vs. 26.8.0%; p = 0.81), live birth rates (36.4% vs. 14.3%; p = 0.46), or perinatal outcomes. In the 209 cycles involving the use of frozen donor sperm, no significant differences were seen between the two groups, except that the mean birth weights were significantly lower with fresh oocyte pregnancies than with warmed oocytes (2952±196 gm vs 2643±700 gm; p = 0.006).ConclusionsEmergent oocyte cryopreservation is a feasible strategy to manage unexpected sperm retrieval failure from severe oligospermic or NOA patients on the oocyte retrieval day. There is no detrimental effect on the live birth rate when testicular spermatozoa or frozen donor sperm are injected into the thawed oocytes compared with fresh oocytes.