Prior exposure to chemotherapy does not reduce the in vitro maturation potential of oocytes obtained from ovarian cortex in cancer patients.

Does chemotherapy exposure affect IVM potential of immature oocytes retrieved from the ovarian cortex following ovarian tissue cryopreservation (OTC) for fertility preservation? The IVM potential of oocyte retrieved from ovarian cortex following OTC is not affected by prior exposure to chemotherapy but primarily dependent on patient's age, while successful retrieval of immature oocytes from the ovarian tissue is negatively affected by chemotherapy and its timing. The potential and feasibility of IVM in premenarche patients was previously demonstrated, in smaller studies. The scarce data that exist on the IVM potential of oocytes retrieved during OTC following chemotherapy support the feasibility of this process, however, this was not previously shown in the premenarche cancer patients population or in larger cohorts. A retrospective cohort study evaluating 229 cancer patients aged 1-39 years with attempted retrieval of oocytes from the ovarian tissue and the medium following OTC in a university affiliated fertility preservation unit between 2002 and 2021. A total of 172 chemotherapy naïve and 57 chemotherapy exposed patients aged 1-39 years underwent OTC in university affiliated tertiary infertility and IVF center. OTC and IVM outcomes were compared between the chemotherapy naïve and exposed groups. The main outcome measure was mean IVM rate per patient in the chemotherapy naïve and exposed groups, with subgroup analysis of a 1:1 chemotherapy exposed group matched for age at OTC and type of malignancy. We additionally analyzed premenarche and postmenarche patients' outcomes separately and investigated the effect of time from chemotherapy to IVM, malignancy type and chemotherapy regimen on oocyte number and IVM outcomes in the chemotherapy exposed group. While the number of retrieved oocytes and percentage of patients with at least one oocyte retrieved was higher in the chemotherapy naïve group (8.7 ± 7.9 versus 4.9 ± 5.6 oocytes and 87.2% versus 73.7%, P < 0.001 and P = 0.016, respectively), IVM rate and number of mature oocytes were comparable between the groups (29.0 ± 25.0% versus 28. 9 ± 29.2% and 2.8 ± 3.1 versus 2.2 ± 2.8, P = 0.979 and P = 0.203, respectively). Similar findings were shown in subgroup analyses for premenarche and postmenarche groups. The only parameter found to be independently associated with IVM rate in a multivariable model was menarche status (F = 8.91, P = 0.004). Logistic regression models similarly showed that past chemotherapy exposure is negatively associated with successful retrieval of oocytes while older age and menarche are predictive of successful IVM. An age and the type of malignancy matched (1:1) chemotherapy naïve and exposed groups were created (25 patients in each group). This comparison demonstrated similar IVM rate (35.4 ± 30.1% versus 31.0 ± 25.2%, P = 0.533) and number of matured oocytes (2.7 ± 3.0. versus 3.0 ± 3.9 oocytes, P = 0.772). Type of malignancy and chemotherapy regimen including alkylating agents were not associated with IVM rate. This study's inherited retrospective design and the long study period carries the possible technological advancement and differences. The chemotherapy exposed group was relatively small and included different age groups. We could only evaluate the potential of the oocytes to reach metaphase II in vitro but not their fertilization potential or clinical outcomes. IVM is feasible even after chemotherapy broadening the fertility preservation options of cancer patients. The use of IVM for fertility preservation, even after exposure to chemotherapy, should be further studied for optimal postchemotherapy timing safety and for the in vitro matured oocytes potential for fertilization. No funding was received for this study by any of the authors. The authors report that no competing interests. N/A.

Karavani G ，Vedder K ，Gutman-Ido E ，Gruda Sussman R ，Goldschmidt N ，Mordechai-Daniel T ，Ben-Meir A ，Imbar T ... -  《-》

Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. N/A.

Abir R ，Ben-Aharon I ，Garor R ，Yaniv I ，Ash S ，Stemmer SM ，Ben-Haroush A ，Freud E ，Kravarusic D ，Sapir O ，Fisch B ... -  《-》

In vitro maturation rates in young premenarche patients.

To evaluate in vitro maturation (IVM) efficacy and oocyte retrieval rates after ovarian tissue cryopreservation in young premenarche girls facing chemo- and radiotherapy. A retrospective cohort study. University-affiliated tertiary medical center. A total of 84 chemotherapy-naïve patients ages 0-18 years referred for fertility preservation between 2004 and 2017: 33 premenarche and 51 postmenarche patients. None. IVM in the pre- and postmenarche groups and in the subgroups of very young (up to age 5 years) and older (5-10 years) premenarche girls. The number of oocytes retrieved did not significantly differ between the postmenarche and premenarche groups (10.8 ± 8.5 and 8.1 ± 6.8, respectively). However, the overall IVM rate was significantly higher in the postmenarche group (28.2% vs. 15.5%, respectively; odds ratio = 0.47). A separate analysis for patients up to 5 years of age demonstrated significantly lower oocyte yield compared with the older (5-10 years) premenarche girls (4.7 ± 5.2 vs.10.3 ± 7.0 oocytes, respectively) and much lower IVM rates (4.9% and 18.2%, respectively). Correlation of age with number of retrieved and matured oocytes showed a positive significant correlation (r = 0.45 and r = 0.64, respectively). IVM performed after ovarian tissue cryopreservation in premenarche girls and specifically in very young girls (4 years and younger) yields substantially decreased maturation rates compared with postmenarche patients, raising a question as to the utility of current IVM technique in this age group. Further studies are required to assess modification of the IVM technique for young girls.

Karavani G ，Schachter-Safrai N ，Revel A ，Mordechai-Daniel T ，Bauman D ，Imbar T ... -  《-》

Combining fertility preservation procedures to spread the eggs across different baskets: a feasibility study.

What is the reproductive potential following combinations of ovarian stimulation, IVM and ovarian tissue cryopreservation (OTC) in female patients seeking fertility preservation (FP)? In selected patients, combining different FP procedures is a feasible approach and reproductive outcomes after FP in patients who return to attempt pregnancy are promising. FP is increasingly performed in fertility clinics but an algorithm to select the most suitable FP procedure according to patient characteristics and available timeframe is currently lacking. Vitrification of mature oocytes (OV) and OTC are most commonly performed, although in some clinical scenarios a combination of procedures including IVM, to spread the sources of gametes, may be considered in order to enhance reproductive options for the future. Retrospective, observational study in a university-based, tertiary fertility centre involving all female patients who underwent urgent medical FP between January 2012 and December 2018. Descriptive analysis of various FP procedures, either stand-alone or combined, was performed, and reproductive outcomes of patients who attempted pregnancy in the follow-up period were recorded. In total, 207 patients underwent medical FP. Patient-tailored strategies and procedures were selected after multidisciplinary discussion. When deemed feasible, FP procedures were combined to cryopreserve different types of reproductive tissue for future use. The main primary outcome measure was the number of mature oocytes. Live birth rates were evaluated in patients who returned for reproductive treatment. Among patients seeking FP, 95/207 (46%) had breast cancer, 43/207 (21%) had haematological malignancies and 31/207 (15%) had a gynaecological tumour. Mean ± SD age was 27.0 ± 8.3 years. Eighty-five (41.1%) patients underwent controlled ovarian stimulation (COS), resulting in 10.8 ± 7.1 metaphase II (MII) oocytes for vitrification. Eleven (5.3%) patients had multiple COS cycles. Transvaginal oocyte retrieval for IVM was performed in 17 (8.2%) patients, yielding 9.2 ± 10.1 MII oocytes. Thirty-four (16.4%) patients underwent OTC combined with IVM of oocytes retrieved from ovarian tissue 'ex vivo' (OTO-IVM), yielding 4.0 ± 4.3 MII oocytes in addition to ovarian fragments. Seventeen (8.2%) patients had OTC combined with OTO-IVM and transvaginal retrieval of oocytes for IVM from the contralateral ovary, resulting in 13.5 ± 9.7 MII oocytes. In 13 (6.3%) patients, OTC with OTO-IVM was followed by controlled stimulation of the contralateral ovary, yielding 11.3 ± 6.6 MII oocytes in total. During the timeframe of the study, 31/207 (15%) patients have returned to the fertility clinic with a desire for pregnancy. Of those, 12 (38.7%) patients had preserved ovarian function and underwent ART treatment with fresh oocytes, resulting in nine (75%) livebirth. The remaining 19 (61.3%) patients requested warming of their cryopreserved material because of ovarian insufficiency. Of those, eight (42.1%) patients had a livebirth, of whom three after OTO-IVM. To date, 5/207 patients (2.4%) achieved an ongoing pregnancy or livebirth after spontaneous conception. Our FP programme is based on a patient-tailored approach rather than based on an efficiency-driven algorithm. The data presented are descriptive, which precludes firm conclusions. Combining different FP procedures is likely to enhance the reproductive fitness of patients undergoing gonadotoxic treatment but further follow-up studies are needed to confirm this. No external funding was used for this study and the authors have no competing interests. N/A.

Delattre S ，Segers I ，Van Moer E ，Drakopoulos P ，Mateizel I ，Enghels L ，Tournaye H ，De Vos M ... -  《-》

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. N/A.

Amargant F ，Zhou LT ，Yuan Y ，Nahar A ，Krisher RL ，Spate LD ，Roberts RM ，Prather RS ，Rowell EE ，Laronda MM ，Duncan FE ... -  《-》