Background/Objectives: Women undergoing fertility preservation by oocyte vitrification before oncological treatments often have a poorer ovarian response to stimulation than healthy women. This indicates that cancer itself can adversely affect ovarian function. However, this impact remains unclear, and its underlying mechanisms are poorly understood. We investigated in this study whether breast cancer, the most common form of cancer in women of reproductive age, alters ovarian function by itself. Methods: For this purpose, we compared the ovarian response to hormonal stimulation in women with breast cancer undergoing oocyte cryopreservation with that in oocyte donors, adjusting for age and BMI. We analysed our data according to the molecular subtype of breast cancer, tumour grade, lymph node invasion, and BRCA1 mutation status. Secondly, we evaluated whether breast cancer alters cholesterol homeostasis in cumulus cells, given its essential role in oocyte quality. The expression of genes involved in cholesterol biosynthesis was analysed in cumulus cells using RT-qPCR, while the concentrations of cholesterol and its intermediates were quantified in follicular fluid using GC-FID and GC-MS/SIM. Results: Compared with oocyte donors, breast cancer patients exhibited a significant decrease in collected oocytes after stimulation. At the molecular level, our data revealed a significant deregulation of cholesterol biosynthesis gene expression in cumulus cells of women with breast cancer. The quantification of cholesterol and its intermediates in follicular fluid revealed altered concentrations in women with breast cancer, suggesting a disrupted follicular microenvironment. Conclusions: These findings suggest that breast cancer impairs ovarian function, at least in part, by disrupting cholesterol homeostasis, which can lead to reduced oocyte competence.

Recent advances in cancer diagnosis and therapy have increased the 5-year survival rate. However, anticancer treatments are gonadotoxic; therefore, fertility preservation is recommended prior to the initiation of therapy to optimize future chances of conceiving. The only method for preserving fertility in prepubescent girls, also suitable for women, is ovarian tissue cryopreservation (OTC). Although approximately 250-300 births have been reported with this approach, significant variability in cryopreservation protocols is observed across fertility preservation centres. Importantly, the presence of residual disease in cryopreserved ovarian fragments will prevent the restoration of fertility. The primary aim of this review is to offer an overview of the impact of cancer and its treatment on female fertility, including the latest updates on novel therapies. This will help to refine fertility preservation indications - a growing concern in the oncofertility field. This review will also provide a comprehensive summary of the different procedures used for OTC, with the aim of standardizing protocols in clinical practice, and offer an updated overview of alternative, albeit experimental, approaches that are being developed to restore fertility and overcome the limitations of transplantation. This will highlight next-generation solutions for restoring the fertility of young female patients.

Elective fertility preservation (EFP) is publicly funded in France, but little data is available about the socio-demographic profiles and motivations of women opting for this procedure, as well as on their perspective and levels of knowledge on the subject. To address this issue, a national multicenter study was conducted within the French CECOS network using an anonymous online questionnaire between November 2023 and June 2024 to women who had started of completed EFP process. A total of 351 responses were recorded. Most women requesting EFP were aged 33 years or older (87.2

OBJECTIVE:This study aimed to evaluate the feasibility of using indocyanine green (ICG) to assess vascularisation by visualizing ovarian fluorescence after laparoscopic cystectomy or plasmajet therapy. The secondary objective was to assess its potential as a surgical marker for predicting the risk of ovarian reserve impairment. SETTINGS:The research design was a prospective feasibility study conducted in the gynecological surgery department at Clermont Ferrand University Hospital. PARTICIPANTS:A total of 45 participants aged 18 to 42 years who required laparoscopic surgical treatment for a benign ovarian cyst (<10 cm in diameter) were included in the study. INTERVENTION:The participants underwent laparoscopic cystectomy or plasmajet therapy with intraoperative evaluation of ovarian vascularisation using ICG fluorescence. Ovarian reserve was assessed using AMH levels and AFC at baseline (M0) and at 6 and 12 months postoperatively (M6 and M12). Pregnancy outcomes were also recorded. RESULTS:Fluorescence was observed in 100% of cases, with Likert scores ≥3 in 80% of the participants. No adverse effects related to ICG were identified. No correlation was found between ICG intensity and ovarian reserve measures. Postoperative follow-up demonstrated a decrease in AMH levels at M6, followed by a halt in this decline at M12 with a tendency toward amelioration halt at M12(2.42 [1.12; 2.71] at M0 then 1.23 [0.37; 2.23] ng/mL at M6; p < 0.001) then 1.46 [0.66; 2.67] ng/mL at M12; p < 0.342) and increased AFC (M0: 11 [8; 18] ; M + 6: 17 [9; 20]; p = 0.018 ; M + 12: 19 [12; 27]; p = 0.001). Among the 26 participants who desire pregnancy, 38.5% conceived spontaneously, and 16% were referred to Assisted Reproductive Technology (ART). CONCLUSION:The use of ICG fluorescence is a feasible approach to the intraoperative assessment of ovarian vascularisation. However, its utility in guiding clinical decisions remains to be demonstrated.

BACKGROUND:Ionizing radiation is widely used in medical diagnostics and cancer therapy, notably radioiodine therapy for thyroid carcinoma. While high or repeated ionizing radiation doses are known to impair male fertility, the impact of clinically relevant single-dose exposures on sperm functional and nuclear integrity is less understood. OBJECTIVES:This study aimed to evaluate the in vitro effects of ionizing radiation doses corresponding to standard therapeutic and diagnostic exposures on human sperm parameters. MATERIALS AND METHODS:Using a computed tomography scan, ejaculated sperm samples (n = 96) were exposed in vitro to 14 mGy of ionizing radiation (equivalent to single abdominal-pelvic computed tomography scan, n = 35), 70 mGy (five computed tomography scans, n = 39), and 140 mGy (one radioiodine therapy with 3.7 GBq of [131I]iodine, n = 86). For each of the three dose conditions, normozoospermic and non-normozoospermic samples (WHO 2021) were included. Functional (vitality, motility) and nuclear biomarkers (chromatin deprotamination, DNA fragmentation, and sperm telomere length), oxidative stress markers (MDA-TBARS), and capacitation parameters (AMPc concentration, phosphotyrosine profiles, velocity, and acrosome integrity) were assessed against paired controls. RESULTS:Exposure to 14, 70, and 140 mGy of ionizing radiation induced significant reductions in sperm vitality (p < 0.01 for 14 mGy and p < 0.001 for 70 and 140 mGy), as well as in total and progressive motility (p < 0.001 for 140 mGy). However, sperm vitality and motility values remained within the WHO 2021 reference ranges. Absorbed doses of 70 and 140 mGy increased deprotamination (p < 0.05 and 0.01, respectively). Effect sizes were mostly small, except for a medium to large effect for vitality at the highest dose. DNA fragmentation, telomere length, oxidative stress, and capacitation-associated markers remained unaffected. These effects were consistent across normozoospermic and non-normozoospermic samples. DISCUSSION AND CONCLUSION:Clinically relevant low-doses of ionizing radiation produced modest impairments in sperm functional and chromatin integrity without detectable DNA breakage or oxidative injury. These findings support the relative safety of low-dose therapeutic exposures on mature spermatozoa. This in vitro model also offers a platform for further exploration of sperm radiosensitivity.

Fertility preservation should be offered to patients facing gonadotoxic therapy. The method for preserving prepubescent girls' fertility, which is also suitable for women, is ovarian tissue cryopreservation (OTC). Although 200 births have been reported worldwide with this approach, significant improvements are needed. The literature indeed reports numerous protocols for freezing and thawing ovarian tissue, with no clear rationale for selection criteria. This study aims to optimize human OTC protocols by characterizing the thermodynamic properties of freezing medium. The freezing medium associated with most live births after autograft (Leibovitz L-15 medium with 4 mg/mL human serum albumin (HSA), 1.5M DMSO, and 0.1M sucrose) was characterized using differential scanning calorimetry. We obtained -120.49 °C for glass transition temperature (Tg'), -20 °C for crystallization temperature when cooling at 2.5 °C/min (Tc) and -4.11 °C for melting temperature (Tm). With these parameters, we optimized a freezing protocol in a programmable freezer (Nano-Digitcool, Cryo Bio System) and a thawing protocol. The freezing curve was as follows: 5 min at 4 °C, 1 °C/min to -7 °C, seeding: 60 °C/min to -32 °C, and 10 °C/min to -15 °C, 0.3 °C/min to -40 °C, 10 °C/min to -140 °C. The thawing protocol consisted in a 3.5-min step in a cold chamber to reach slowly Tg', limiting thermal and mechanical shocks, and then a 2-min incubation at 37 °C to quickly reach Tm. Ovarian tissue frozen-thawed according to these protocols had a similar quality to that of fresh tissue and could resume folliculogenesis during organotypic culture. Our study will contribute to improve human OTC and optimize women fertility preservation.

Objective: To study karyotypes of >8,200 oocyte donor candidates in nulliparous or multiparous women compared with a reference population. Design: A retrospective observational multicentric study. Subjects: The study included 2 cohorts of oocyte donor candidates recruited between January 2005 and October 2021: multiparous women with at least 1 child at the time of recruitment and nulliparous women. Both were compared with a reference population composed of female newborns from literature. Exposure: Not applicable. Main Outcome Measures: Blood lymphocyte karyotype. Results: A total of 8,229 oocyte donor candidates from 22 fertility centers were included in this study. Nulliparous (n = 1,890) and multiparous (n = 6,339) women were compared with 8,102 female newborns. Overall, 65 candidates were carriers of chromosomal abnormalities and were, therefore, excluded from the donation process (0.79%; 95% confidence interval [CI], 0.60-0.98). The occurrence of balanced structural chromosomal rearrangements globally increased in the study population (0.49%; 95% CI, 0.34-0.64) compared with that in female newborns (0.24%; 95% CI, 0.34-0.64). The number of reciprocal translocations increased fivefold in nulliparous oocyte donor candidates (0.37%; 95% CI, 0.10-0.64). The incidence of sex chromosome mosaicism notably increased in multiparous oocyte donor candidates, with 17 cases (0.27%; 95% CI, 0.14-0.40). Among chromosomal aberration carriers, only 2 nulliparous women (1 reciprocal translocation and 1 sex chromosome mosaicism) had fertility issues with a diagnosis of premature ovarian failure. Conclusion: In this comprehensive 16-year French experience of karyotyping in oocyte donor candidates, we confirmed an increased incidence of balanced structural chromosomal rearrangements, especially among those without children at the time of recruitment. Karyotyping could be considered to identify any chromosomal abnormalities that may not be easily detectable through medical questioning. These abnormalities pose an inherent genetic risk for gamete recipients if left undetected. (Fertil Steril (R) 2025;123: 692-9. (c) 2024 by American Society for Reproductive Medicine.) El resumen est & aacute; disponible en Espa & ntilde;ol al final del art & iacute;culo.

BACKGROUND:Numerous publications have questioned the lack of analytical reliability and clinical relevance of sperm morphology assessment for infertility workup and before use of assisted reproductive techniques (ART). There is a huge variability in the performance and interpretation of this test. It has become necessary to evaluate its true medical service rendered to the patient. OBJECTIVES:To develop clinical guidelines for use of spermatozoa morphology assessment during male fertility check-up and before ART. MATERIALS AND METHODS:These guidelines were produced following a pre-defined standard methodology for narrative and Patient Intervention Comparison Outcomes (PICO) questions. The French Working Group (WG) on Sperm Morphology Assessment consisted of 15 members including an expert in statistics. RESULTS:R1: WG does not recommend systematic detailed analysis of abnormalities (or groups of abnormalities) during sperm morphology assessment. R2: WG recommends that the laboratory should use a qualitative or quantitative method for detection of a monomorphic abnormality (globozoospermia, macrocephalic spermatozoa syndrome, pinhead spermatozoa syndrome, multiple flagellar abnormalities). The result may be given as an interpretative commentary or as a numerical report of the percentage of detailed abnormalities. R3: There is insufficient evidence to demonstrate the clinical value of indexes of multiple sperm defects (TZI, SDI, MAI) in investigation of infertility and before ART. Accordingly, the working group does not recommend the use of sperm abnormality indexes (TZI, SDI, MAI) in sperm morphology assessment. R4: WG gives a positive opinion on the use of automated systems based on cytological analysis after staining after qualification of the operators, and validation of the analytical performance within their own laboratory. R5: WG does not recommend using the percentage of spermatozoa with normal morphology as a prognostic criterion before IUI, IVF, or ICSI, or as a tool for selecting the ART procedure. DISCUSSION:This article examines the clinical interest of sperm morphology assessment during fertility check-up and before ART. The overall level of evidence from studies is low, challenging current practices regarding sperm morphology assessment. CONCLUSION:These guidelines suggest a significant simplification of sperm morphology assessment in the light of the examined publications while maintaining the detection of monomorphic sperm abnormalities.

Study question Do ionizing radiations affect human sperm integrity? If so, at what dose? Summary answer In vitro exposure of human ejaculated spermatozoa significantly reduces vitality, motility, and chromatin integrity without causing DNA fragmentation, telomeric damage, or altering capacitation ability. What is known already Exposure to ionizing radiation (IR) is a public health concern. However, its impact on human spermatozoa remains poorly described, particularly regarding nuclear damage. Our objective is to assess the impact of IR on functional parameters, as well as on nuclear quality. To achieve this, we expose human spermatozoa in vitro to increase doses of IR, reflecting various diagnostic and therapeutic medical scenarios. This study will help elucidate the mechanisms by which IR affects male fertility and, consequently, optimize fertility preservation strategies. Study design, size, duration We exposed human spermatozoa in vitro to increase IR doses mimicking epididymis irradiation during medical procedure: diagnostic imaging (scanner CT: 14–70 mGy) and radioiodine therapy (140 mGy). Sperm vitality, motility, DNA fragmentation, chromatin decondensation, telomere length (STL), nuclear morphometry, oxidative stress and capacitation markers post-IR were compared to their non-irradiated controls for each patient to address inter-individual variability. Participants/materials, setting, methods We used surplus semen from patient having consented to research (Germethèque Biobank). Gamma radiation conditions mimicking CT doses of 14 (n = 35), 70 (n = 39), and 140 mGy (n = 73) were experimentally calibrated. Sperm parameters (WHO 2021 guidelines), DNA fragmentation (TUNEL/SCD assays), chromatin decondensation (Chromomycin A3), STL (qPCR), nuclear morphometry (3D bioimage analysis), oxidation (Oxiselect/MDA kits) and capacitation markers -velocity (SCA© CASA), [AMPc] (ELISA), phosphotyrosine profile (Western blot) and acrosome integrity (PSA-FITC)- were evaluated. Main results and the role of chance The three dose groups showed no significant differences in initial sperm parameters or bio-clinical criteria, confirming the absence of biases. We observed a significant decrease in sperm vitality across all three radiation doses compared to the non-irradiated control condition (-3.4% for 14 mGy, -4.2% for 70 mGy, and -5.6% for 140 mGy; p < 0.001). Progressive motility was reduced at 140 mGy (37.0 ± 2.3% versus (vs.) 33.1 ± 2.0% after IR exposure; -3.9%, p < 0.001). IR induced chromatin decondensation at 70 mGy (23.6 ± 1.9% vs. 26.0 ± 1.8%, +2.3%, p < 0.05) and 140 mGy (23.5 ± 1.3% vs. 26.1 ± 1.5%, +2.5%, p < 0.01), with greater decondensation observed in samples with pre-existing sperm parameter alterations (+3.5%, p < 0.05). No significant changes were detected in DNA fragmentation levels (TUNEL or SCD assays) or sperm telomere length (STL). Exposure to 140 mGy did not significantly alter oxidative levels in seminal fluid or sperm cells, nor did it affect capacitation markers, although motility alterations were identified using SCA®. Finally, no significant global impact on 3D nuclear morphometric parameters has been observed to date. Limitations, reasons for caution This model provides an opportunity to analyze IR-induced alterations without patients/pathology’s interference. Differences in dose rate, exposure duration, and the spermatozoa environment (seminal instead of epididymal fluid) represented limitations. In vivo studies are essential to fully understand the impacts of IR on the regulation pathways of spermatogenesis and sperm maturation. Wider implications of the findings We show for the first time the impact of IR on human sperm quality using three radiations doses used for clinical diagnosis or treatment. Significant but minor effects on conventional parameters and chromatin decondensation were observed. Ongoing work includes epigenomic alterations from sperm cells and seminal fluid to further investigate. Trial registration number Yes

BACKGROUND:Perm freezing compromises nuclear integrity. Standardized tools to assess three-dimensional (3D) chromatin alterations are lacking. OBJECTIVES:This study aimed to present a novel protocol for 3D nuclear morphometric analysis of human spermatozoa to measure slow freezing and thawing-induced alterations. MATERIALS AND METHODS:Human sperm cells were examined before and after freezing using a consistent 3D nuclear morphometric analysis protocol that distinguishes between live and dead spermatozoa. Morphometric assessments were performed by microscopy and image analysis with the NucleusJ2.0/NODeJ software, without introducing denaturing agents. The analysis included measurements of nuclear volume, elongation, flatness, and the volume of hypercondensed chromatin (Hc) regions, along with the relative fluorescence intensity of these zones (RHF intensity). Additional parameters evaluated comprised sperm vitality, motility, DNA fragmentation, and chromatin decondensation. RESULTS:A quantitative analysis of 4919 sperm nuclei from 10 patients demonstrated significant modifications in the hypercondensed chromatin (Hc) zones, with a marked decrease in sperm vitality and motility (p  <  0.001) and a significant increase in DNA fragmentation (p  <  0.05). At the chromatin level, slow freezing induced a higher number of Hc zones per nucleus (p  <  0.01), a reduction in the average volume of these zones (p  <  0.0001), and a reduced relative fluorescence intensity (p  <  0.01). Notably, these chromatin alterations were most pronounced in viable spermatozoa. DISCUSSION AND CONCLUSION:This study is the first to provide standardized 3D nuclear morphometry measurements for human sperm, offering a novel biomarker to assess male fertility and cryopreservation susceptibility, with potential clinical applications for improving patient care. Slow freeze-thaw cycle induced significant alterations in sperm chromatin, disrupting nuclear organization and forming several smaller, less compacted hypercondensed chromatin zones.

BACKGROUND:No study has yet explored telomere length or interaction in sperm nuclei of testicular cancer (TC) patients exposed to chemotherapy or radiotherapy. However, sperm telomere dynamics have emerged as a potential marker in male infertility. OBJECTIVES:We aimed to investigate in a pilot and exploratory study whether TC and its adjuvant treatments alter sperm telomeres and DNA integrity during a 2-year follow-up after treatment. MATERIALS AND METHODS:This ancillary study is part of the multicentric prospective and longitudinal French GAMATOX study. Twenty-nine TC patients treated with orchiectomy and either adjuvant chemotherapy (n = 14) or radiotherapy (n = 15) were included, alongside ten fertile donors. Semen samples were collected before orchiectomy and adjuvant therapy, and at 3, 6, 12, and 24 months after treatment. Sperm telomere length (STL) and telomere interactions were assessed by quantitative fluorescent in situ hybridisation (FISH). Sperm DNA fragmentation (SDF) was measured using the TUNEL assay, aneuploidy by FISH, and chromatin condensation defects by aniline blue staining. RESULTS:Before adjuvant therapy, patients presented a higher number of sperm telomere signals compared with controls (p = 0.004), persisting 2 years after treatment (p = 0.001). STL was not affected by tumour histology. Extremely short telomeres and the highest number of telomere signals per nucleus were more frequently observed after chemotherapy. SDF and chromatin condensation defects were transient and paralleled the recovery of conventional semen parameters, regardless of treatment type. A moderate increase in sperm aneuploidy was observed 6 months after chemotherapy (p = 0.03). CONCLUSION:TC patients presented impaired sperm telomere interactions before and after adjuvant therapy, as reflected by a persistently elevated number of telomere signals per nucleus, and a higher prevalence of extremely short sperm telomeres, especially after chemotherapy. While relative STL remained comparable to controls, these findings raise questions about the impact of telomere architecture on germ cell tumour biology and post-treatment fertility.

Gonadotropin injections used to stimulate oocyte production during assisted reproductive technology (ART) procedures are associated with the risk of an abnormal response in predisposed patients suffering polycystic ovary syndrome (PCOS). Liver X receptors (LXR) pathway has been identified as key regulators during this process. This study explores the integration of the hormonal signals, cellular networks and molecular mechanisms linking sterol signaling with inflammation and immune infiltration. Pharmacological activation of LXR in a wild-type context protects against gonadotropin hyperstimulation mirroring the effect observed in LXR-deficient mice. Ovarian stimulation leads to immune cell infiltration orchestrated by granulosa cells in absence of LXR, resulting in an altered granulosa cell response to gonadotropin and enhanced inflammation. LXR controls inflammasome activity by regulating Thioredoxin Interacting Protein (TXNIP) gene expression in mural granulosa cells, thereby modulating IL1β production. This immune cell infiltration persists throughout ovulation in PCOS patients and is observed in cumulus oocytes complexes, highlighting the pivotal role of LXR path in regulating inflammatory processes during hormonal stimulation in ART procedures.

Are the 3D morphometry parameters of the human sperm nucleus affected by slow freezing? The slow freezing-thawing cycle induces significant alterations on hypercondensed nucleus zone by reducing its volume and splits it into several small zones. Sperm cryopreservation is an essential technique for infertility care, but it seems to highly affect the genetic and epigenetic integrity of sperm nuclei with poorly identified mechanisms. Moreover, it is known that sperm nuclear alterations may be involved in pregnancy failures. Therefore, sperm nuclear biomarkers are an essential issue to assess the outcome of ART procedures when frozen sperm is used. Thereby, 3D visualization of chromatin is a good way to understand modifications in the organization and composition of sperm chromatin after a slow freezing procedure. Ten semen of patients were analyzed (Germetheque biobank) after informed consent. Standard semen parameters, DNA fragmentation, chromatin decondensation, sperm telomere length (STL) and 3D nuclear morphometry were performed before and after slow freezing. Morphometric measurements were performed from a digital representation of the sperm nucleus in 3 dimensions that allow us to measure the volume, flatness and elongation of each sperm nucleus and characterize hypercondensed zones, with live/dead spermatozoa distinction. After addition of cryoprotective medium v/v (Cryosperm®), sperm was frozen with a programmable freezer (NanoDigitcool®). Standards sperm parameters were measured in accordance with WHO guidelines 2021, DNA fragmentation by flow cytometry TUNEL assay, chromatin decondensation by epifluorescent microscopy with Chromomycin A3 and STL using qPCR. 3D nuclear representation was performed by a z-axis image acquisition series of DAPI/PI stained slides with optigrid epifluorescent microscopy. Analyses were carried out using the NucleusJ2.0 software with NODeJ plugin. Freezing induces an alteration of sperm vitality (p < 0.0001) and progressive and total motility (p < 0.001 and p < 0.0001 respectively) and an increase in the percentage of sperm with DNA fragmentation (18.30% fresh vs 31.6% frozen, p < 0.05). We did not observe a modification of the median volume of the sperm nucleus before and after freezing (37.15 [IC = 36.5;38.14] and 37.10 [33.27;37.78] µm3 respectively), as the same for flatness and elongation (2.48 vs. 2.50 a.u. and 2.04 vs. 2.04 a.u. respectively). Nevertheless, five samples showed significant alterations of nucleus volume (p < 0.0001), due to diminution of flatness and more or less elongated. A significant decrease of the total volume of the hypercondensed chromatin zone was observed after freezing (0.94 [0.84;0.99] before vs. 0.61 [0.53;0.69] µm3 after freezing-thawing, p < 0.0001), with an effect size of -1.48 [-2.10; -0.86]. Decrease of total volume of hypercondensed region was measured homogeny in all the samples tested, particularly on live (1.00 vs. 0.68 µm3 respectively, p < 0.0001) compared with dead spermatozoa (0.72 vs. 0.57 µm3, p < 0.05). We showed also a significant increase of the number of hypercondensed zones in each nucleus (p < 0.001), reflecting a split into several parts of the hypercondensed chromatin. A 3D-FISH analysis would allow to characterize the hypercondensed chromatin portion, probably as heterochromatin and which we suspect to be the nucleus ring. Moreover, a chromosomes specific marking would allow for the identification of whether whole chromosomes or specific parts of chromosomes (centromeres or telomeres) are affected by this decompaction. This study is the first to make exacts measurements of human sperm nucleus because of a non-decondensing method. We demonstrate that slow freezing affects hypercondensed chromatin probably through a decondensation and modification of the nucleus-specific organization. This new assessment will allow a better proficiency of men fertility preservation techniques. NCT04715828

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16

STUDY QUESTION Is large for gestational age (LGA) observed in babies born after frozen embryo transfer (FET) associated with either the freezing technique or the endometrial preparation protocol? SUMMARY ANSWER Artificial cycles are associated with a higher risk of LGA, with no difference in rate between the two freezing techniques (vitrification versus slow freezing) or embryo stage (cleaved embryo versus blastocyst). WHAT IS KNOWN ALREADY Several studies have compared neonatal outcomes after fresh embryo transfer (ET) and FET and shown that FET is associated with improved neonatal outcomes, including reduced risks of preterm birth, low birthweight, and small for gestational age (SGA), when compared with fresh ET. However, these studies also revealed an increased risk of LGA after FET. The underlying pathophysiology of this increased risk remains unclear; parental infertility, laboratory procedures (including embryo culture conditions and freezing-thawing processes), and endometrial preparation treatments might be involved. STUDY DESIGN, SIZE, DURATION A multicentre epidemiological data study was performed through a retrospective analysis of the standardized individual clinical records of the French national register of IVF from 2014 to 2018, including single deliveries resulting from fresh ET or FET that were prospectively collected in fertility centres. Complementary data were collected from the participating fertility centres and included the vitrification media and devices, and the endometrial preparation protocols. PARTICIPANTS/MATERIALS, SETTING, METHODS Data were collected from 35 French ART centres, leading to the inclusion of a total of 72 789 fresh ET, 10 602 slow-freezing FET, and 39 062 vitrification FET. Main clinical outcomes were presented according to origin of the transferred embryos (fresh, slow frozen, or vitrified embryos) and endometrial preparations for FET (ovulatory or artificial cycles), comparing five different groups (fresh, slow freezing-ovulatory cycle, slow freezing-artificial cycle, vitrification-ovulatory cycle, and vitrification-artificial cycle). Foetal growth disorders were defined in live-born singletons according to gestational age and sex-specific weight percentile distribution: SGA and LGA if <10th and >= 90th percentiles, respectively. Analyses were performed using linear mixed models with the ART centres as random effect. MAIN RESULTS AND THE ROLE OF CHANCE Transfers led to, respectively, 19 006, 1798, and 9195 deliveries corresponding to delivery rates per transfer of 26.1%, 17.0%, and 23.5% after fresh ET, slow-freezing FET, and vitrification FET, respectively. FET cycles were performed in either ovulatory cycles (n = 21 704) or artificial cycles (n = 34 237), leading to 5910 and 10 322 pregnancies, respectively, and corresponding to pregnancy rates per transfer of 31.6% and 33.3%. A significantly higher rate of spontaneous miscarriage was observed in artificial cycles when compared with ovulatory cycles (33.3% versus 21.4%, P < 0.001, in slow freezing groups and 31.6% versus 21.8%, P < 0.001 in vitrification groups). Consequently, a lower delivery rate per transfer was observed in artificial cycles compared with ovulatory cycles both in slow freezing and vitrification groups (15.5% versus 18.9%, P < 0.001 and 22.8% versus 24.9%, P < 0.001, respectively). Among a total of 26 585 live-born singletons, 16 413 babies were born from fresh ET, 1644 from slow-freezing FET, and 8528 from vitrification FET. Birthweight was significantly higher in the FET groups than in the fresh ET group, with no difference between the two freezing techniques. Likewise, LGA rates were higher and SGA rates were lower in the FET groups compared with the fresh ET group whatever the method used for embryo freezing. In a multivariable analysis, the risk of LGA following FET was significantly increased in artificial compared with ovulatory cycles. In contrast, the risk of LGA was not associated with either the freezing procedure (vitrification versus slow freezing) or the embryo stage (cleaved embryo versus blastocyst) at freezing. Regarding the vitrification method, the risk of LGA was not associated with either the vitrification medium used or the embryo stage. LIMITATIONS, REASONS FOR CAUTION No data were available on maternal context, such as parity, BMI, infertility cause, or maternal comorbidities, in the French national database. In particular, we cannot exclude that the increased risk of LGA observed following FET with artificial cycles may, at least partially, be associated with a confounding effect of some maternal factors. No information about embryo culture and incubation conditions was available. Most of the vitrification techniques were performed using the same device and with two main vitrification media, limiting the validity of a comparison of risk for LGA according to the device or vitrification media used. WIDER IMPLICATIONS OF THE FINDINGS Our results seem reassuring, since no potential foetal growth disorders following embryo vitrification in comparison with slow freezing were observed. Even if other factors are involved, the endometrial preparation treatment seems to have the greatest impact on LGA risk following FET. FET during ovulatory cycles could minimize the risk for foetal growth disorders. STUDY FUNDING/COMPETING INTEREST(S) This work has received funding from the French Biomedicine Agency (Grant number: 19AMP002). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

The COVID-19 pandemic, driven by SARS-CoV-2, led authorities to recommend halting assisted reproductive technology programs, focusing instead on fertility preservation, for cancer patients. The presence of SARS-CoV-2 in semen remains controversial. This multicentric prospective cohort study, conducted across 12 university medical centers, aimed to determine if SARS-CoV-2 is present in spermatozoa/seminal plasma in cancer patients by RT-PCR and to assess its impact on standard semen parameters. The levels of cytokines and TNF-alpha were measured in seminal fluid by ELISA. We enrolled 129 men who underwent sperm cryopreservation between July 7, 2020, and June 30, 2021. The 63 were included and tested for COVID-19 in nasal swab samples by RT-PCR and/or by serology. All patients were asymptomatic on the day of semen collection: 50 were uninfected, 8 had a positive nasal swab (PCR+) and 5 were seropositive. SARS-CoV-2 RNA was not detected in the seminal fluid or spermatozoa. Ejaculate volume was significantly lower in the PCR+ group compared to the uninfected group (median [IQR]: 2.6 mL [1.6-3.4] vs. 4.6 mL [2.6-5.2] p < 0.05). Total and progressive motility were lower in the PCR+ group compared to the seropositive group (32.5% [25.0-45.0] vs. 50% [49.0-55.0] p < 0.05, and 22.5% [10.0; 32.5] vs. 44.5% [40-49] p < 0.05). Higher TNF-alpha level was observed in the PCR+ group (1.9 pg/mL [0-3.9]) compared to the uninfected group (0 pg/mL [0-0.4]) p < 0.05. Although SARS-CoV-2 was not detected in the sperm samples of cancer patients who were PCR+, the infection appears to impact sperm parameters, likely due to inflammation.

Abstract Study question Does the presence of breast cancer alter the ovarian response to hormonal stimulation and the cholesterol homeostasis in ovarian follicles? Summary answer The deleterious impact of breast cancer on the ovarian response might be link to an alteration of the cholesterol biosynthesis in cumulus cells. What is known already Breast cancer is the most frequent cancer in reproductive-aged women. The cancer itself seems to exert a deleterious impact on the ovarian functions. Indeed, cancer patients who undergo fertility preservation before cancer therapy initiation show a poorer response to ovarian stimulation than healthy women. Nevertheless, this impact remains controversial, and the mechanisms by which the cancer impairs the ovarian functions are poorly understood. To our knowledge, no study has yet evaluated the impact of breast cancer on the cholesterol biosynthesis pathway in human cumulus cells, a pathway essential for oocyte competence to maturation and fertilization. Study design, size, duration 30 patients with breast cancer (10 triple negative (TNBC), 10 hormone-receptor positive (HR+) and 10 HER2 positive (HER2+)) and 30 women oocytes donors undergoing oocyte cryopreservation will be included from January 2019. We assess ovarian response to stimulation (number, quality of retrieved oocytes; total FSH dose). We quantify the level of enzymes and regulators of the cholesterol biosynthesis pathway in cumulus cells (CC) by RT-qPCR. We plan to quantify cholesterol levels in follicular fluids (FF). Participants/materials, setting, methods Since January 2019, 25 breast cancer patients (9 TNBC, 8 HR+ and 8 HER2+) and 30 oocytes donors (control group) under 37 years old have been included. Ovarian stimulation was performed with antagonist protocol. Following ovarian pick up and denudation, CC and FF were collected. After total RNAs extraction from CC, the expression levels of enzymes and regulators of the cholesterol biosynthesis pathway were quantified by RT-qPCR. Cholesterol levels will be quantified in FF. Main results and the role of chance The clinico-biological characteristics (age, BMI) are comparable between cancer patients and the control group (p > 0.05). Serum AMH levels are lower in the TNBC subgroup (2.3±1.6 ng/mL) than in the control group (4.7±2.8 ng/mL, p = 0.04). A poorer ovarian response to ovarian stimulation is observed in the breast cancer group compared with the control group (number of mature oocytes: 9.4±5.2 vs. 14.5±7.7, p = 0.007). Among the cancer patients group, the TNBC subgroup shows the poorest ovarian response to stimulation (number of mature oocytes: 7.6±4.3 vs. 14.5±7.7 in control group, p = 0.006) despite the highest total administered FSH dose (2480±880 IU vs. 1831±460 IU in control group, p = 0.02). Dysregulation of the cholesterol biosynthesis pathway is observed in the breast cancer group. For TNBC and HR+ cancer patients, we observed an increase in the transcripts levels of CYP51 enzyme (respectively 3.28±1.69 vs. 1.84±2.33, p = 0.0098 and 1.77±1.78 vs. 1.84±2.33, p = 0.03) and SREBP2 activator (respectively 2.04±0.95 vs. 1.27±1.13, p = 0.003 and 1.12±0.47 vs. 1.27±1.13, p = 0.02) of the cholesterol biosynthesis pathway compared to the control group. In HER2+ cancer patients, a significant decrease in the expression levels of SQLE (0.39±0.34 vs 1.81±3.99, p = 0.001) and DHRC7 enzymes (0.33±0.32 vs 1.21±0.88, p = 0.0002) is observed compared to the control group. Limitations, reasons for caution Our preliminary results should be confirmed with the analysis of a larger number of participants. We should confirm that the observed modulations in the expression levels of enzymes and regulators of the cholesterol biosynthesis pathway led to a loss of cholesterol homeostasis by quantifying cholesterol in FF. Wider implications of the findings Our findings strengthen the hypothesis that cancer exerts a deleterious impact on the ovarian functions, as we observed a lower ovarian response in patients with breast cancer. This project will have clinical implications, such as to adapt the fertility preservation approach according to the breast cancer subtype. Trial registration number Not applicable