STUDY QUESTION Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.
Study question Could controlled ovarian stimulation (COS) protocols used in fertility preservation (FP) impact on malignant cell proliferation and tumour molecular profiling of breast cancer (BC) patients? Summary answer Letrozole supplementation during ovarian stimulation for oocyte vitrification could be considered as a safe procedure in estrogen-dependent BC patients undergoing FP. What is known already High estradiol levels associated to COS could promote changes in gene expression in estrogen-positive BC tumors. Estradiol levels reached during the ovarian stimulation could aggressively promote malignant cell proliferation and cell migration to adjacent organs. Aromatase inhibitors such as letrozole, are added to standard stimulation protocols to avoid this undesirable potential side effect. Despite the reassuring clinical results achieved by using letrozole for FP in BC patients, there is still a lack of evidence regarding its impact on malignant cell behaviour. For this reason, specific molecular studies to properly evaluate safety of letrozole in this specific population are still required. Study design, size, duration Experimental in vivo study. Thirty 5-week-old Nude-nu female mice were divided into three different groups: BC (n = 10), BC and FSH stimulation (BC-FSH, n = 10), or BC and letrozole stimulation (BC-LTZ, n = 10). BC was considered the control group, whereas BC-FSH and BC-LTZ represented distinct COS protocols. Hormone-dependent BC was induced in all mice. Animals were followed-up for 5 months and then euthanized to collect kidney, ovary, spleen, and liver tissues for gene expression and immunohistochemistry (IHC) analysis. Participants/materials, setting, methods One million of human MCF–7 BC cells were injected into the mouse left kidney capsule. Two days after xenograft, COS was induced by 10IU FSH or 1mg/ml letrozole + 10IU FSH, followed by ovarian triggering with 10IU hCG at 48h. Human BC RT2 Profiler PCR Arrays were performed to evaluate the impact of COS on tumour behaviour. BC biomarkers (Ki67, Erα, PR and HER–2) were also analyzed by IHC to validate gene expression results. Main results and the role of chance The differential gene expression was firstly assessed in kidney samples, as they represent the xenograft site, and different expression profiles were obtained depending on the COS protocol used. The BC-FSH group showed a global over-expression pattern of all genes of the array when compared to BC and BC-LTZ. Further gene ontology analysis revealed that cellular process, biological regulation, metabolic process, and proteases were the most over-represented biological terms, with a 20.5-fold over-expression for MMP2 compared to the other groups. On the other hand, BC-LTZ mice presented gene expression profiles similar to that of controls. When other tissues were analysed to detect malignant cell presence, our results revealed a significant up-regulation of matrix-proteases, cell cycle and proliferation related-genes, in liver samples from the BC-FSH group, but no amplification of any of the studied genes was detected in ovarian tissue or spleen. IHC findings confirmed the presence of human BC cells in 100% of samples from kidney tissue and in 30% of samples from liver tissue in the BC-FSH group. No human cells were detected by IHC in the BC and BC-LTZ groups. Limitations, reasons for caution Since this is an animal model of estrogen-dependent BC induced through a cell line, further validation with human tumour breast cancer samples would be required. Wider implications of the findings: Adjuvant letrozole in COS protocols prevents BC cell migration. The present study suggests that this protective effect could be mediated by interfering ER-pathway downstream genes involved in cell proliferation and matrix digestion. Altogether, letrozole could safely be used as a supplement during COS procedures for oocyte vitrification in BC women. Trial registration number Not applicable
Study question What is the impact of fertility preservation (FP) procedures and cancer treatment on relapse, survival, ovarian damage and pregnancy outcomes in oncological patients? Summary answer FP technique (including ovarian stimulation for oocyte vitrification) does not affect relapse or survival rates even in hormone dependant tumours. What is known already FP has become a crucial part of oncological evaluation of young women facing cancer given the high survival rates achieved. Oocyte vitrification (OV) and ovarian cortex cryopreservation (OCC) are the main techniques offered to these patients at the moment. However, information on the true incidence of premature ovarian insufficiency (POI), return rates to use the cryopreserved material and the natural pregnancy rates achieved in these patients is still limited. Moreover although there is some data about the safety of these techniques the impact of FP on disease survival is yet to be definitely assessed. Study design, size, duration Prospective cohort study. 695 patients enrolled since 2001 until 2016. Patients referred to FP unit in a public hospital setting (Hospital Peset Valencia 2001-2006 and University Hospital La Fe 2007-2016). After evaluation 556 patients received a FP technique (OV, OCC or embryo vitrification ) and 139 patients did not receive any due to medical reasons or patient’s choice. Minimum follow-up 5 years after enrolment Participants/materials, setting, methods Baseline characteristics including type of cancer and previous chemotherapy at diagnosis and prior to FP technique were recorded followed by risk of chemotherapy treatment received, relapse, survival, POI and poor ovarian reserve (POR) occurrence and pregnancy outcomes. Primary outcome was median survival time after FP in months. Secondary outcomes included relapse rate, POI and POR incidence, usage FP rate, clinical pregnancy and live birth (LB) rates naturally and after FP use. Main results and the role of chance There were no differences in survival comparing patients undergoing FP versus no FP (median 89.67 vs 92.81 months, p = 0.3). However, patients that used their cryopreserved material survived more than those who did not (97.3 vs 89.5, p = 0.012). When assessing survival rates comparing patients that had approval to get pregnant versus those who did not we found a higher survival in the former (98.84 vs 84.79 months, p < 0.001). Breast cancer patients with hormone dependent tumors undergoing ovarian stimulation for OV vs OCC had no differences in survival (95.62 vs 87.38 months, p = 0.37). POI incidence was 20.29% (N = 141). POI patients were significantly older (32.28 vs 29.63, p < 0.001) and had received high-risk chemotherapy more frequently (31.74% vs 2.27%, p < 0.001). Ovarian damage incidence (including also POR) was 48.06% (N = 334). Eighty-six patients (15.47%) used their cryopreserved material. Among the patients with pregnancy wish (N = 266) there were 84 spontaneous live births (31.58%). Patients that conceived naturally were significantly younger (30.71 vs 33.46, p < 0.001) and the chemotherapy received was more frequently low-risk (43.20% vs 23.52%, p = 0.018). There were 37 LB after use of FP (37/86, 43.02%). Patients with a higher ovarian reserve percentile had a higher chance of achieving a natural LBR (OR 1.016, 1.005-1.027, p = 0.004) Limitations, reasons for caution The higher survival found in patients using their cryopreserved material is mediated by the prognosis of the disease itself that limits the chance of pregnancy to those with a stable disease. Wider implications of the findings FP does not have a negative impact on survival even if ovarian stimulation is used. Almost half of the patients had ovarian damage (POI or POR) as a result of treatment; this is higher than previously reported. Among the patients with fertility wish around one third achieved a LB naturally. Trial registration number not applicable
Study question Does the D19S884 allele 8 (A8) equally affect the pathogenesis and ovarian gene expression of PCOS and PCOM patients? Summary answer A8-allele produces metabolic, endocrine, and ovarian alterations regardless diagnose. The mechanisms involved in PCOM alterations are different from those of the ovarian phenotype of PCOS. What is known already The specific D19S884 allele 8 of the FBN3 gene may be related to polycystic ovarian syndrome (PCOS) clinical manifestations. The A8 allele participates in alternative splicing of FBN3 and produces Asprosin-3, related to glucose modulation, and Fibrillin-3. Fibrilin-3 is an extracellular matrix protein, and together with a dysregulation of the Hippo pathway, could be responsible for constraining follicular growth in the PCOS ovaries. However, it is still unknown how these pathways act in PCOS, and whether these mechanisms are also involved in pathophysiology of polycystic ovarian morphology (PCOM) in apparently normal women with regular menses. Study design, size, duration Cross-sectional and descriptive study with 139 women (24-39 years-old) undergoing an IVF cycle between 2019-2022 at Hospital La Fe (Valencia, Spain). Thirty-one patients were considered PCOS, twenty-eight were classified as PCOM while the remaining eighty were controls. After recruitment women were screened for A8 allele, metabolic status, hormone profile, follicular fluid protein determination and expression of Hippo pathway and extracellular matrix genes. The IVF cycle parameters were recorded to determine their relationship with study variables. Participants/materials, setting, methods Women with two or more Rotterdam-criteria were considered as PCOS. PCOM patients were defined by polycystic ovarian morphology on ultrasound with regular ovulatory cycles. Genomic DNA was isolated from blood samples to assess the presence of A8 allele by capillary electrophoresis and FBN3 concentration was measured on follicular fluid by ELISA. TaqMan qPCR assay was used to analyze the expression of Hippo pathway (BIRC1 and CCN2) and extracellular matrix (ECM) genes in cumulus cells. Main results and the role of chance PCOS patients showed statistically significant metabolic and endocrine alterations with higher BMI, free androgen index (FAI), and glucose levels compared to controls. Despite having increased AMH levels (PCOS:45.0±21.7; PCOM:33.8±21.5; Control:17.6±7.19pmol/L, p < 0.05), and AMH/AFC ratio (PCOS:1.9±0.9; Control:1.2±0.5, p = 0.008), PCOS showed lower fertilization rates (PCOS:64±26; Control:80±18%, p = 0.008), reduced good quality (PCOS:0.6±1.0; Control:1.3±1, p = 0.003) and transferred embryos (PCOS:1.3±0.9; Control:1.9±1.0, p = 0.018). In PCOM, metabolic and androgen profiles were not different from controls. Although AMH, AFC (PCOM:24.4±13.7; Control:15.5±7.1, p = 0.007) and aspirated follicles (PCOM:17.3±5.7; Control:14.3±7.0, p = 0.026) were increased, fewer embryos were transferred (PCOM:1.2±1.1; Control:1.9±1.0, p = 0.027). PCOM women also showed ovarian downregulation of the Hippo-pathway (CCN2-PCOM:-4.8±10.8; Control:0.1±3.0, p = 0.010) and increased FBN3 concentration (PCOM:19.6±8.8; Control:14.7±5.6ng/ml, p=N.S). The A8 allele was detected in 17% of our patients: 4% PCOS, 50% PCOM and 46% controls. Overall, A8 presence associated a Hippo-pathway downregulation (BIRC1-A8+:-2.7±4.5; A8-:0.3±2.2, p = 0.034) and increased ECM expression (EMILIN1-A8+:2.1±1.0; A8-:1.2±1.5, p = 0.04). Interestingly, in controls, higher glucose (A8+:95.1±6.7; A8-:83.5±10.1mg/dl, p = 0.002), cholesterol (A8+:182.5±11.1; A8-:165.4±27.3mg/dl, p = 0.029), LDL (A8+:117.8±10.4; A8-:83.9±29.4mg/dl, p = 0.002), and DHEAS levels (A8+:2633.0±670.7; A8-:1585.8±670.0ng/ml, p = 0.026) were found, consistent with a reduction in HDL (A8+:51.8±8.8; A8-:67.4±15.43mg/dl, p = 0.026) and a downregulation of the Hippo-pathway (BIRC1-A8+:-4.8±4.6; A8-:0.2±4.6, p = 0.013). In PCOM, A8+ promoted higher FAI (A8+:1.6±1.5; A8-:0.8±0.7, p=N.S), and less embryos obtained (A8+:5.9±3.8; A8-:10.7±3.8, p = 0.04). Limitations, reasons for caution Due to the low prevalence of A8 in our PCOS population, its effects cannot be evaluated in this group. Further validation of gene expression results by RNA-seq will be required to assess the broad spectrum of ovarian transcriptomic changes induced by both, the PCOM phenotype and the A8 allele. Wider implications of the findings Polycystic ovarian morphology is not unique to clinical disorder of PCOS and the triggering mechanisms appear to be distinct from those observed in women with regular cycles. Our findings suggest that the presence of A8 allele impacts on metabolic profile, androgen levels, and ovarian pathway dysregulations, despite the clinical diagnose. Trial registration number Not applicable
Context: Some vaginal lubricants and ultrasound gels are known to be detrimental to sperm function and therefore could negatively affect fertility. Aims: The aim of the current study was to develop a sperm motility index (SMI) to test the sperm toxicity of ultrasound gels and vaginal lubricants used in reproductive medicine. Settings and Design: Two ultrasound gels (Aquasonic ® and Kefus ® ) and five vaginal lubricants (Vaginesil™, Velastisa ® , K-Y Jelly ® , Control ® , and Durex ® ) were studied. Three different concentrations (1%, 5%, and 10%) of each lubricant were tested. Subjects and Methods: SMI was calculated dividing the percentage of progressively motile sperm in each tested gel by that in the control at 0.5, 1, 2, and 24 h of incubation at 5% of CO 2 and 37°C. SMI values <0.75 indicate sperm toxicity. Statistical Analysis Used: The main outcome measured was SMI for each concentration and time of incubation. Results: Only Durex ® did not show any deleterious effect on sperm quality. The rest of lubricants presented different degrees of toxicity. Vaginesil™ resulted in toxic for all concentrations and incubation periods (SMI < 0.12). Control ® and Velastisa ® presented toxicity at 10% after 2 h, while K-Y Jelly ® showed toxicity at 10% from 1 h of incubation. Regarding ultrasound gels, Aquasonic ® showed toxic effects after only 0.5 h (SMI = 0.70 ± 0.15), while Kefus ® showed slightly toxic effects after 2 h (SMI 0.69 ± 0.07). Conclusions: SMI is an accurate tool to evaluate sperm toxicity. One of the main strengths of the article is the inclusion of representative semen samples and known products used worldwide. This study has a relevant clinical translation since it highlights the importance of evaluating the possible sperm toxicity of simple products used in reproductive medicine.
Study question Could controlled ovarian stimulation (COS) protocols with letrozole supplementation induce changes in the molecular footprints of estrogen receptor-positive (ER+) breast cancer (BC) women? Summary answer Ovarian stimulation with letrozole and gonadotropins does not change the gene expression profile of estrogen receptor-positive (ER+) breast malignant tumours as compared to non-stimulated patients. What is known already Fertility preservation (FP) strategies in ER+ BC patients involve modifications of the classical COS protocols to minimise the impact of estrogen levels during ovarian stimulation. The most common approaches are using the aromatase inhibitor letrozole to block the conversion of testosterone into estradiol in the granulosa cells or the use of tamoxifen to block the ER in the breast tissue. Although retrospective studies suggest that these strategies will have no impact on patient’s survival and disease progression, little is known regarding the effect of the ovarian stimulation on the genomic fingerprints of the exposed cancerous tissue. Study design, size, duration Retrospective, non-randomized, comparative study. Gene expression profiles were compared in biopsies at diagnosis and during excision surgery in two groups of ER+ BC patients: Patients undergoing COS for FP before surgery (COS group, n = 10) and patients not undergoing any type of COS (Control group, n = 11). The former received letrozole (5mg/day) supplements during COS. The latter included 7 patients not doing FP and 4 undergoing ovarian cortex cryopreservation. Patients were recruited between 2009 and 2021. Participants/materials, setting, methods Single 5-µm sections of formalin-fixed, paraffin-embedded (FFPE) needle core biopsy (NCB) and breast surgery (BS) tumour samples were obtained prior to starting gonadotoxic therapies. Differential gene expression and gene set analysis (GSA) was performed in tumour samples before (NCB-sample) and after COS (BS-sample). Simultaneous, quantitative detection of 2549 genes associated with tumour biology was performed with the HTG EdgeSeq Oncology Biomarker Panel. Tumour cell proliferation was also assessed by Ki67 staining. Main results and the role of chance Patients were younger in the COS group (COS: 30.2±3.4 vs Control: 34.5±2.3; p = 0.004). None of the patients experienced any relapse during the observation period. The length of COS was 11.5±3.5 days and 10.6±6.8 MII oocytes were vitrified. The exploratory analysis using principal component analysis revealed no relationships between the two BC biopsy samples and gene expression levels in the experimental groups. The differential expression analysis just revealed 6 genes significantly over-expressed after ovarian stimulation (DUSP1, FOS, EGFR1, NR4A1, JUN and CYR61). From these, DUSP1, FOS, and NR4A1 were also significantly upregulated in the unstimulated group. The GSA showed the cytochrome P450 pathway was significantly enriched after COS. No pathways related to cell proliferation were differentially expressed between groups. However, in unstimulated patients, 6 KEGG pathways were upregulated, with the cytokine-cytokine receptor interaction and the Jak-STAT signalling pathway being the most enriched. Ki67 immunostaining showed no differences in cell proliferation after COS (22.8±7.2 vs 24.0±6.9, p = 0.723). The rate of cell proliferation also remained constant in unstimulated patients (22.9±8.4 vs 25.1±8.1, p = 0.560). Limitations, reasons for caution The main limitations of the present study are the retrospective design and the associated risk of bias, although the population is very homogeneous when it comes to clinical characteristics; and the fact that molecular footprints are only interim markers of survival/response to the treatment. Wider implications of the findings The present study provides molecular evidence supporting the safety of COS using letrozole for oocyte vitrification in young BC patients undergoing ovarian stimulation. This information is crucial to support patient guidance during discussions about fertility preservation. Trial registration number Not applicable
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