Single-cell analysis comparing early-stage oocytes from fresh and slow-frozen/thawed human ovarian cortex reveals minimal impact of cryopreservation on the oocyte transcriptome.

Does the slow-freezing and thawing process have a negative impact on the transcriptome of oocytes isolated from early-stage human follicles compared to fresh controls? The transcriptional profiles of fresh and frozen/thawed oocytes did not cluster separately, indicating undetectable differences between the two groups when compared to within-donor heterogeneity. Previous studies using histological analysis of follicle morphology, density, and stage distribution in slow-frozen/thawed human ovarian cortex compared to fresh controls showed no differences between the two groups. Clinical cases reported in the past 10 years have demonstrated that transplanted slow-frozen/thawed and fresh ovarian cortex restored normal serum FSH levels and regular menstrual cycles by 5 months. However, the slow-frozen and thawed tissue resulted in lower rates of pregnancies and live births, albeit not statistically significant. We utilized single-cell RNA-sequencing (scRNAseq) of 144 human oocytes isolated from cadaver ovaries obtained from three donors. Human ovarian cortex from three healthy premenopausal donors 16, 18, and 27 years old was cut into squares measuring 10 × 10 × 1 mm3 and either slow-frozen and thawed or processed fresh. First, using a novel method for isolating live oocytes from primordial and primary follicles, the ovarian cortex squares were fragmented with a McIlwain tissue chopper and enzymatically digested. Next, oocytes were mechanically denuded under a dissection microscope and placed individually into wells containing lysis buffer for scRNAseq. Lysed single oocytes were subjected to library prep using the seqWell PlexWell rapid single-cell RNA protocol. Pooled libraries were subjected to 150-bp paired-end sequencing on the NovaSeq6000 Illumina platform. In total, we sequenced 144 oocytes-24 oocytes isolated fresh and 24 oocytes isolated after slow-freezing and thawing from each of the three donors. Additionally, we performed histological analysis of fresh and frozen/thawed ovarian cortex tissue from all three donors using hematoxylin and eosin staining and analyzed morphology, follicle density, and follicle stage distribution differences between fresh and cryopreserved ovarian cortex. The histological analysis revealed no differences in follicle stage distribution or follicle morphology between conditions, with the percentage of normal follicles in fresh and frozen/thawed tissue, respectively, as 86.7% and 91.0% for Donor 1, 91.7% and 92.5% for Donor 2, and 96.1% and 91.1% for Donor 3. The follicle density per mm3 in fresh and frozen/thawed tissue, respectively, was 279.4 and 235.8 for Donor 1, 662.2 and 553.5 for Donor 2, and 55.8 and 71.4 for Donor 3. The difference in follicle density was not statistically significant between fresh and frozen/thawed conditions for Donors 2 and 3, and significant (P = 0.017) for Donor 1. The stromal cell densities in fresh and frozen/thawed tissue, respectively, were 0.014 in both conditions for Donor 1, 0.014 and 0.016 for Donor 2, and 0.013 and 0.014 for Donor 3. There was no statistically significant difference in stromal cell density between conditions in Donor 1 and Donor 3, though it was statistically significant (P ≤ 0.001) for Donor 2. The transcriptional profiles of fresh and frozen/thawed oocytes did not cluster separately, suggesting insignificant differences between the two groups. However, at the group mean level, there was a small shift between the fresh and frozen/thawed oocytes and the shifts were parallel across the three donors. In this comparison, fresh oocytes were enriched for gene ontology terms related to chromosome segregation and mitosis, whereas frozen/thawed oocytes were enriched for terms related to wound response, cAMP signaling, and extracellular matrix organization. Datasets available on Zenodo.org. DOI: https://zenodo.org/records/13224872. In this study, we only sequenced the oocytes isolated from early-stage follicles due to technical challenges collecting and sequencing the somatic cells surrounding the oocytes. Investigating the transcriptomic changes after freezing and thawing in the somatic cells would need to be studied in the future. Additionally, we built RNAseq libraries immediately after thawing focusing on the immediate changes. Investigation of the effects that manifest at later timepoints, either in culture or upon implantation in an animal model, may reveal additional effects of the freeze/thaw process on the transcriptome. The only clinically approved method of fertility preservation for prepubertal cancer patients and adult patients who cannot delay cancer treatment is ovarian tissue cryopreservation. Investigation of cryopreservation-induced changes in follicles at all stages is critical to further our understanding of the safety and efficacy of using these tissues for fertility preservation in the clinic. Our study is the first to analyze transcriptomic changes between individual fresh and slow-frozen/thawed human oocytes collected from early-stage follicles. To accomplish this, we developed a novel method for dissociating both fresh and frozen/thawed human ovarian cortex to obtain live denuded oocytes from early-stage follicles. Our findings provide insights into the use of cryopreserved tissue and follicles for fertility preservation efforts. This work was funded by National Institutes of Health (NIH) R01HD099402, Career Training in Reproductive Biology (CTRB) Training Grant National Institutes of Health (NIH) T32 to Jordan Machlin, National Institutes of Health (NIH) F31-HD106626 and National Institutes of Health (NIH) T31H-D079342 to Andrea Jones, National Institutes of Health (NIH) T32-GM70449 to D. Ford Hannum, and The Chan Zuckerberg Initiative Grant CZF2019-002428. We have no conflicts of interest to declare.

Machlin JH ，Hannum DF ，Jones ASK ，Schissel T ，Potocsky K ，Marsh EE ，Hammoud S ，Padmanabhan V ，Li JZ ，Shikanov A ... -  《-》

In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. The number of different donors used was limited. Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. N/A.

Cheng H ，Wei F ，Del Valle JS ，Stolk THR ，Huirne JA ，Asseler JD ，Pilgram GSK ，Van Der Westerlaken LAJ ，Van Mello NM ，Chuva De Sousa Lopes SM ... -  《-》

Comparison of fresh testicular sperm aspiration and use of either thawed pre-frozen sperm or oocyte freezing: impact on cumulative live birth rates for couples experiencing ejaculation failure.

Is there a difference in the cumulative live birth rate (CLBR) after fresh testicular sperm aspiration (TESA) compared with the use of either pre-frozen sperm or oocyte freezing for couples experiencing ejaculation failure on the day of oocyte retrieval? After adjusting for confounding factors, the use of pre-frozen sperm or the freezing and thawing of oocytes appeared to be as effective as TESA in achieving CLBRs for couples experiencing temporary ejaculation failure. Male patients may be concerned about experiencing temporary ejaculation failure on the day of their partner's oocyte retrieval, in which case they may choose surgical sperm retrieval, oocyte freezing on the day, or have their sperm frozen in advance. However, the clinical efficacy of these three options has not yet been evaluated. A retrospective data analysis was conducted on 65 178 oocyte retrieval cycles at a university-affiliated IVF center from January 2012 to May 2021. The overall characteristics, completed cycle characteristics, and clinical outcomes were analyzed among couples with ejaculation failure who underwent three distinct clinical interventions, with those receiving TESA serving as the control group. The primary outcome measure was the CLBR, and the secondary outcome measures were the clinical pregnancy rate (CPR) and live birth rate (LBR) per embryo transfer. A robust (modified) Poisson regression model was used to evaluate the association between the three clinical options for ejaculation failure and CLBRs. Of the eligible oocyte retrieval cycles, 756 cycles (1.2%) experienced ejaculation failure, with 640 cycles completing treatment. These treatments included 325 cycles using TESA, 227 cycles utilizing pre-frozen sperm, and 88 cycles involving frozen-thawed oocytes. The CLBRs for the TESA, thawed-sperm and thawed-oocyte groups were 36.9%, 48.9%, and 34.1%, respectively, showing a statistically significant difference (P = 0.007). Specifically, the thawed-sperm group demonstrated a significantly higher CLBR compared to the TESA group, while no significant difference was observed between the TESA and thawed-oocyte groups. Similarly, the CPRs and LBRs per embryo transfer for the three groups were 37.4%, 50.0%, and 41.8%, respectively (P = 0.005), and 29.9%, 39.6%, and 33.0%, respectively (P = 0.030). Again, the thawed-sperm group showed a significantly higher CPR and a significantly higher LBR, but no significant differences for the thawed-oocyte group, compared to the TESA group. Notably, the significant differences in both CLBR and LBR emerged after the second embryo transfer. However, after adjusting for multiple factors, including female age at oocyte retrieval, type and duration of infertility, female body mass index, number of previous IVF cycles, ovarian stimulation protocol, endometrial thickness on the last ultrasound, insemination method, number of oocytes retrieved, number of fertilized oocytes, and number of usable embryos on Day 3, the analysis revealed no significant association between CLBR and the use of pre-frozen sperm (risk ratio (RR) 1.08, 95% confidence interval (CI) 0.81-1.44) or thawed oocytes (RR 1.01, 95% CI 0.76-1.33), compared with TESA. Given that the study is retrospective and the sample size is too small, particularly concerning the use of thawed oocytes, we acknowledge that the data present here is only suggestive and refers to an association that warrants cautious interpretation. Therefore, further research in the form of prospective studies as well as randomized controlled trials is needed to provide a definitive answer to the research question. Our findings suggest that using pre-frozen sperm or frozen-thawed oocytes can offer comparable CLBRs to TESA for cases of temporary ejaculation failure, providing clinical alternatives that may reduce the logistical challenges in ART cycles. This study was supported by the National Nature Science Foundation of China (grant nos. 82101672, 82171589), the National Key Research and Development Program of China (grant nos. 2022YFC2702504, 2019YFE0109500), the Basic and Applied Basic Research Foundation of Guangdong Province (grant no. 2021A1515010774), and the Guangzhou Municipal Science and Technology Project (grant nos. 202102010075, 2023A4J0578). The authors declare that they have no conflict of interest in relation to the data in this paper. N/A.

Zhan S ，An G ，Gan J ，Du H ，Fu X ，Wang C ，Mao Y ，Kang X ，Liu J ，Liu H ... -  《-》

Rapamycin prevents cyclophosphamide-induced ovarian follicular loss and potentially inhibits tumour proliferation in a breast cancer xenograft mouse model.

To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model? Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation. Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation. To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry. Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group. None. The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens. Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients. This work was not financially supported. The authors declare that they have no conflict of interest.

Tanaka Y ，Amano T ，Nakamura A ，Yoshino F ，Takebayashi A ，Takahashi A ，Yamanaka H ，Inatomi A ，Hanada T ，Yoneoka Y ，Tsuji S ，Murakami T ... -  《-》

Outcomes of female fertility preservation with cryopreservation of oocytes or embryos in the Netherlands: a population-based study.

What are the reproductive outcomes of patients who cryopreserved oocytes or embryos in the context of fertility preservation in the Netherlands? This study shows that after a 10-year follow-up period, the utilization rate to attempt pregnancy using cryopreserved oocytes or embryos was 25.5% and the cumulative live birth rate after embryo transfer was 34.6% per patient. Fertility preservation by freezing oocytes or embryos is an established treatment for women with a risk of premature ovarian failure (caused by a benign or oncological disease) or physiological age-related fertility decline. Little is known about the success of cryopreservation, the utilization rate of oocytes or embryos, or the live birth rates. A retrospective observational study was performed in the Netherlands. Data were collected between 2017 and 2019 from 1112 women who cryopreserved oocytes or embryos more than 2 years ago in the context of fertility preservation in 10 IVF centers in the Netherlands. A total of 1112 women were included in this study. Medical files and patient databases were used to extract data. Women were categorized based on indication of fertility preservation: oncological, benign, or non-medical. To indicate statistical differences the t-test or Mann-Whitney U test was used. Kaplan-Meier analyses were used for time endpoints, and log-rank analyses were used to assess statistical differences. The study protocol was approved by the medical ethics committee. Fertility preservation cycles have been performed increasingly over the years in the Netherlands. In the first years, less than 10 cycles per year were performed, increasing to more than 300 cycles per year 10 years later. Initially, embryos were frozen in the context of fertility preservation. In later years, cryopreservation of oocytes became the standard approach. Cryopreservation of oocytes versus embryos resulted in comparable numbers of used embryos (median of 2) for transfer and comparable live birth rates (33.9% and 34.6%, respectively). The 5-year utilization rate was 12.3% and the 10-year utilization rate was 25.5%. The cumulative clinical pregnancy rate was 35.6% and the cumulative live birth rate was 34.6% per patient. Those who had fertility preservation due to benign diseases returned earlier to use their cryopreserved embryos or oocytes. The follow-up period after the fertility preservation procedure varied between patients in this study and not all frozen oocytes or embryos had been used at the end of this study. This might have led to underestimated outcomes reported in this study. Furthermore, intention to treat cannot be fully determined since women who started the fertility preservation procedure without success (cancellation due to low response) were not included in this study. This study provides data on the reproductive outcomes after various indications of fertility preservation. This knowledge can be informative for professionals and future patients to improve counseling and informed decision making regarding ovarian stimulation in the context of fertility preservation. No funding was obtained for this study. The authors have no conflicts of interest to declare related to this study. V.T.H. received grants paid to the institute for studies outside the present work from AstraZeneca, Gilead, Novartis, Eli Lily, Pfizer, and Daiichi Sankyo. V.T.H. received consulting fees from Eli Lily outside the present work. M.G. received grants paid to the institute for studies outside the present work from Guerbet and Ferring. E.M.E.B. received a grant from The Dutch Network of Fertility Preservation for a study outside the present work. N/A.

Ter Welle-Butalid ME ，Derhaag JG ，van Bree BE ，Vriens IJH ，Goddijn M ，Balkenende EME ，Beerendonk CCM ，Bos AME ，Homminga I ，Benneheij SH ，van Os HC ，Smeenk JMJ ，Verhoeven MO ，van Bavel CCAW ，Tjan-Heijnen VCG ，van Golde RJT ... -  《-》