Metabolic signature of follicular fluid in infertility-related diseases: a narrative review.

Metabolomics offers new methods to identify biomarkers for oocyte and embryo quality, and for a better understanding of the physiopathology of infertility. This review investigated the latest findings regarding metabolome-derived biomarkers in follicular fluid of women with the most common types of infertility, and the potential impact on reproductive medicine outcomes. PubMed was searched for publications on metabolomics and human follicular fluid, and key biomarkers, kinetics and relationships with infertility diseases were identified. A reduced concentration of glucose and increased concentrations of lactate and pyruvate were found in follicular fluid of patients with endometriosis and diminished ovarian reserve, and the opposite was found in patients with polycystic ovary syndrome. These signatures may lead to the hypothesis of changed metabolite concentrations in patients with endometriosis and diminished ovarian reserve, and a metabolic pathway alteration with decreased aerobic glycolysis in patients with polycystic ovary syndrome. However, the pattern found in patients with endometriosis and low responders may also be expected in follicular fluid of fertile women. Larger studies are needed to confirm the results. An international database may help to highlight follicular fluid biomarkers in order to improve the selection of cryopreserved oocytes, and to enrich culture medium to restore normal metabolism and improve reproductive treatment outcomes.

When to cryopreserve ovarian tissue: Determining the effects of chemotherapy on the ovarian reserve by studying follicular density and apoptosis.

OBJECTIVES: Patients scheduled to receive chemotherapy should be counselled on fertility preservation. Known gonadotoxic chemotherapies such as alkylating agents have a high risk of altering ovarian reserve. In some cases, the urgency of treatment requires the use of chemotherapy before fertility preservation, which will be carried out at a later stage. Usually the ovarian tissue is cryopreserved. The aim of our study is to investigate the impact of chemotherapies on follicular density and the apoptosis of reserve follicles. METHODS: We included 140 patients: 63 patients, mean age 18.8 years, were included in the group "no chemotherapy" (group A) and 77 patients, mean age 17.1 years, in the group "received chemotherapy before ovarian conservation" (group B). None of the patients had had pelvic radiotherapy prior to ovarian cryopreservation. The histological parameters studied were follicular density and the presence of malignant cells. We selected 12 patients from group A and 15 patients from group B, comparable in age and pathology, for whom we evaluated follicle apoptosis by immunostaining cleaved caspase-3. RESULTS: We demonstrated an inverse relationship between follicular density and age (p < 0.0001), as well as a lack of effect of chemotherapy on follicular density (p = 0.87). We showed the impact of various chemotherapies, especially with alkylating agents, on the apoptosis of ovarian follicles (p < 0.0001). Three patients had ovarian tissue infiltration, two of which were malignant. CONCLUSION: This work underlines the fact that conservation of ovarian tissue after chemotherapy remains possible.

Delaying testicular sperm extraction in 47,XXY Klinefelter patients does not impair the sperm retrieval rate, and AMH levels are higher when TESE is positive.

STUDY QUESTION: Should testicular sperm extraction (TESE) in non-mosaic 47,XXY Klinefelter syndrome (KS) patients be performed soon after puberty or could it be delayed until adulthood? SUMMARY ANSWER: The difference in sperm retrieval rate (SRR) in TESE was not significant between the 'Young' (15-22 years old) cohort and the 'Adult' (23-43 years old) cohort of non-mosaic KS patients recruited prospectively in parallel. WHAT IS KNOWN ALREADY: Several studies have tried to define predictive factors for TESE outcome in non-mosaic KS patients, with very heterogeneous results. Some authors have found that age was a pejorative factor and recommended performing TESE soon after puberty. To date, no predictive factors have been unanimously recognized to guide clinicians in deciding to perform TESE in azoospermic KS patients. STUDY DESIGN, SIZE, DURATION: Two cohorts (Young: 15-22 years old; Adult: 23-43 years old) were included prospectively in parallel. A total of 157 non-mosaic 47,XXY KS patients were included between 2010 and 2020 in the reproductive medicine department of the University Hospital of Lyon, France. However 31 patients gave up before TESE, four had cryptozoospermia and three did not have a valid hormone assessment; these were excluded from this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data for 119 patients (61 Young and 58 Adult) were analyzed. All of these patients had clinical, hormonal and seminal evaluation before conventional TESE (c-TESE). MAIN RESULTS AND THE ROLE OF CHANCE: The global SRR was 45.4%. SRRs were not significantly different between the two age groups: Young SRR=49.2%, Adult SRR = 41.4%; P = 0.393. Anti-Müllerian hormone (AMH) and inhibin B were significantly higher in the Young group (AMH: P = 0.001, Inhibin B: P < 0.001), and also higher in patients with a positive TESE than in those with a negative TESE (AMH: P = 0.001, Inhibin B: P = 0.036). The other factors did not differ between age groups or according to TESE outcome. AMH had a better predictive value than inhibin B. SRRs were significantly higher in the upper quartile of AMH plasma levels than in the lower quartile (or in cases with AMH plasma level below the quantification limit): 67.7% versus 28.9% in the whole population (P = 0.001), 60% versus 20% in the Young group (P = 0.025) and 71.4% versus 33.3% in the Adult group (P = 0.018). LIMITATIONS, REASONS FOR CAUTION: c-TESE was performed in the whole study; we cannot rule out the possibility of different results if microsurgical TESE had been performed. Because of the limited sensitivity of inhibin B and AMH assays, a large number of patients had values lower than the quantification limits, preventing the definition a threshold below which negative TESE can be predicted. WIDER IMPLICATIONS OF THE FINDINGS: In contrast to some studies, age did not appear as a pejorative factor when comparing patients 15-22 and 23-44 years of age. Improved accuracy of inhibin B and AMH assays in the future might still allow discrimination of patients with persistent foci of spermatogenesis and guide clinician decision-making and patient information. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a grant from the French Ministry of Health D50621 (Programme Hospitalier de Recherche Clinical Régional 2008). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: NCT01918280.

Shallow artificial networks with morphokinetic time-lapse parameters coupled to ART data allow to predict live birth.

Purpose: The purpose of this work was to construct shallow neural networks (SNN) using time-lapse technology (TLT) from morphokinetic parameters coupled to assisted reproductive technology (ART) parameters in order to assist the choice of embryo(s) to be transferred with the highest probability of achieving a live birth (LB). Methods: A retrospective observational single-center study was performed, 654 cycles were included. Three SNN: multilayers perceptron (MLP), simple recurrent neuronal network (simple RNN) and long short term memory RNN (LSTM-RNN) were trained with K-fold cross-validation to avoid sampling bias. The predictive power of SNNs was measured using performance scores as AUC (area under curve), accuracy, precision, Recall and F1 score. Results: In the training data group, MLP and simple RNN provide the best performance scores; however, all AUCs were above 0.8. In the validating data group, all networks were equivalent with no performance scores difference and all AUC values were above 0.8. Conclusion: Coupling morphokinetic parameters with ART parameters allows to SNNs to predict the probability of LB, and all SNNs seems to be efficient according to the performance scores. An automatic time recognition system coupled to one of these SNNs could allow a complete automation to choose the blastocyst(s) to be transferred.

Development and Survival of Human Ovarian Cells in Chitosan Hydrogel Micro-Bioreactor.

Background and Objectives: To test the long-term ability of human ovarian cortex cells to develop in unconventional culture conditions. Materials and Methods. Ovarian cortex cells from fetuses aged 23 to 39 weeks gestation were cultured for 90 days in hollow chitosan hydrogel micro-bioreactors and concurrently in traditional wells. Various cell-type counts were considered. Results: With intact follicles as a denominator, the percentage of growing intact follicles at Day 0 varied widely between ovaries (0 to 31.7%). This percentage tended to increase or stay relatively constant in bioreactor as in control cultures; it tended more toward an increase over time in bioreactor vs. control cultures. Modeled percentages showed differences (though not significant) in favor of bioreactor cultures (16.12% difference at D50 but only 0.12% difference at D90). With all follicles present as a denominator, the percentage of growing primary and secondary follicles at D0 varied widely between ovaries (0 to 29.3%). This percentage tended to increase over time in bioreactor cultures but to decrease in control cultures. Modeled percentages showed significant differences in favor of bioreactor cultures (8.9% difference at D50 and 11.1% difference at D90). At D50 and D90, there were only few and sparse apoptotic cells in bioreactor cultures vs. no apoptotic cells in control cultures. Conclusions: Over three months, bioreactor folliculogenesis outperformed slightly traditional culture. This is an interesting perspective for follicle preservation and long-term toxicological studies.

ICSI using testicular spermatozoa after failure of ICSI with ejaculated spermatozoa could be a good choice: A propensity score-matched cohort study.

BACKGROUND: Ejaculated spermatozoa are considered to possess a higher fertilisation potential than testicular spermatozoa. In selected cases, the use of testicular spermatozoa from non-azoospermic infertile men resulted in a higher implantation and pregnancy rate than the use of ejaculated spermatozoa. OBJECTIVE: The primary objective was to compare the live birth rate and cumulative live birth rate between couples with failed intracytoplasmic sperm injection procedure using ejaculated spermatozoa who subsequently had an intracytoplasmic sperm injection cycle with testicular spermatozoa and those who subsequently had an intracytoplasmic sperm injection cycle with ejaculated spermatozoa. The secondary objective was to determine the indications for the use of testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa. MATERIALS AND METHODS: A retrospective study of matched couples using propensity score matching analysis was performed. After an intracytoplasmic sperm injection failure (cycle_1), intracytoplasmic sperm injection with either ejaculated spermatozoa (ejaculated sperm group), or testicular spermatozoa (testicular sperm group), was performed (cycle_2). The matching was on intracytoplasmic sperm injection performed in cycle_1 according to spermatozoa used (testicular or ejaculated) in cycle_2. Logistic regression was used to evaluate the influence of sperm origin on cumulative live birth rate. Univariate analysis on parameters of cycle_1 was used to identify the prognostic factors to propose an intracytoplasmic sperm injection with testicular spermatozoa in case of cycle_1 failure. The study outcomes were live birth rate and cumulative live birth rate. RESULTS: Among the 6034 couples available, 63 were selected to constitute the testicular sperm group and 63 were selected by propensity score matching to constitute the ejaculated sperm group. After matching, the DNA fragmentation index was higher in the testicular sperm group (13.43% ± 9.65% vs. 8.93% ± 4.47%, p = 0.013); no significant difference was observed for the fertilisation rate, the number of obtained embryos, blastulation rate and frozen embryo rate. In cycle_2, the live birth rate was higher in the testicular group (22.2% vs. 0.0%, p < 0.001), as was the cumulative live birth rate (25.4% vs. 6.3%, p = 0.065). The prognostic factors identified for the proposal of intracytoplasmic sperm injection procedure with testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa were: teratozoospermia, cryptozoospermia and high DNA fragmentation index. DISCUSSION: According to the present study and current knowledge, the use of testicular spermatozoa after failed intracytoplasmic sperm injection procedure in non-azoospermic men could be proposed instead of sperm donation in case of high sperm DNA fragmentation index, cryptozoospermia and teratozoospermia. A good oocyte response to ovarian stimulation during the previous assisted reproductive technology attempt will increase the chance of success. Although the main limitation of the current study is its retrospective nature, the use of the propensity score matching to perform causal inference study increases its reliability. CONCLUSION: The present study supports that the use of testicular spermatozoa outside the classical indication of azoospermia is a good option when the indication is well established. However, before proposing a testicular biopsy, an improvement in sperm characteristics should be considered by treating the causes of sperm alteration.

A real-world study of ART in France (REOLA) comparing a biosimilar rFSH against the originator according to rFSH starting dose.

BACKGROUND: Since the first launch of a biosimilar recombinant follicle stimulating hormone (rFSH), Bemfola®, in Europe in 2014, it has been possible to study in routine clinical care throughout France the effectiveness of a biosimilar rFSH including according to different rFSH starting doses. METHODS: REOLA was a non-interventional, retrospective, real world study using anonymized data from 17 Assisted Reproductive Technology (ART) centres' data management systems across France including 2,319 ART ovarian stimulation cycles with Bemfola® and 4,287 ART ovarian stimulation cycles with Gonal-f®. For both products, four populations were studied according to starting dose of rFSH: < 150 IU, 150 - 224 IU, 225 - 299 IU and ≥ 300 IU. The primary endpoint was the cumulative live birth rate (cLBR) per commenced ART ovarian stimulation cycle including all subsequent fresh and frozen-thawed embryo transfers starting during a follow up period of at least 1 year following oocyte retrieval. RESULTS: A direct relationship of increasing rFSH starting dose with increasing age, increasing basal FSH, decreasing AMH and increasing body mass index was noted. No clinically relevant differences were seen in all outcomes reported, including the cLBR, between Bemfola® and Gonal-f®, but for both drugs, an association was seen with increasing rFSH starting dose and decreasing cLBR. CONCLUSIONS: The REOLA study demonstrates that the cLBR with Bemfola® is very similar to Gonal-f® across all patient subpopulations. The cLBR is inversely related to the rFSH starting dose irrespective of the drug used, and the REOLA study provides reassurance of the clinical effectiveness of a biosimilar rFSH used in a real world setting.

Update on the management of endometriosis-associated pain in France.

The French National College of Obstetricians and Gynecologists (CNGOF) published guidelines for managing endometriosis-associated pain in 2018. Given the development of new pharmacological therapies and a review that was published in 2021, most national and international guidelines now suggest a new therapeutic approach. In addition, a novel validated screening method based on patient questionnaires and analysis of 109-miRNA saliva signatures, which combines biomarkers and artificial intelligence, opens up new avenues for overcoming diagnostic challenges in patients with pelvic pain and for avoiding laparoscopic surgery when sonography and MRI are not conclusive. Dienogest (DNG) 2 mg has been a reimbursable healthcare expense in France since 2020, and, according to recent studies, it is at least as effective as combined hormonal contraception (CHC) and can be used as an alternative to CHC for first-line treatment of endometriosis-associated pain. Since 2018, the literature concerning the use of DNG has grown considerably, and the French guidelines should be modified accordingly. The levonorgestrel intrauterine system (LNG IUS) and other available progestins per os, including DNG, or the subcutaneous implant, can be offered as first-line therapy, gonadotropin-releasing hormone (GnRH) agonists with add-back therapy (ABT) as second-line therapy. Oral GnRH antagonists are promising new medical treatments for women with endometriosis-associated pain. They competitively bind to GnRH receptors in the anterior pituitary, preventing native GnRH from binding to GnRH receptors and from stimulating the secretion of luteinizing hormone and follicle-stimulating hormone. Consequently, estradiol and progesterone production is reduced. Oral GnRH antagonists will soon be on the market in France. Given their mode of action, their efficacy is comparable to that of GnRH agonists, with the advantage of oral administration and rapid action with no flare-up effect. Combination therapy with ABT is likely to allow long-term treatment with minimal impact on bone mass. GnRH antagonists with ABT may thus be offered as second-line treatment as an alternative to GnRH agonists with ABT. This article presents an update on the management of endometriosis-associated pain in women who do not have an immediate desire for pregnancy.

Treatment algorithms for high responders: What we can learn from randomized controlled trials, real-world data and models.

A high ovarian response to conventional ovarian stimulation (OS) is characterized by an increased number of follicles and/or oocytes compared with a normal response (10-15 oocytes retrieved). According to current definitions, a high response can be diagnosed before oocyte pick-up when >18-20 follicles ≥11-12 mm are observed on the day of ovulation triggering; high response can be diagnosed after oocyte pick-up when >18-20 oocytes have been retrieved. Women with a high response are also at high risk of early ovarian hyper-stimulation syndrome (OHSS)/or late OHSS after fresh embryo transfers. Women at risk of high response can be diagnosed before stimulation based on several indices, including ovarian reserve markers (anti-Müllerian hormone [AMH] and antral follicle count [AFC], with cutoff values indicative of a high response in patients with PCOS of >3.4 ng/mL for AMH and >24 for AFC). Owing to the high proportion of high responders who are at the risk of developing OHSS (up to 30%), this educational article provides a framework for the identification and management of patients who fall into this category. The risk of high response can be greatly reduced through appropriate management, such as individualized choice of the gonadotropin starting dose, dose adjustment based on hormonal and ultrasound monitoring during OS, the choice of down-regulation protocol and ovulation trigger, and the choice between fresh or elective frozen embryo transfer. Appropriate management strategies still need to be defined for women who are predicted to have a high response and those who have an unexpected high response after starting treatment.

An Update on In Vitro Folliculogenesis: A New Technique for Post-Cancer Fertility.

INTRODUCTION: Obtaining in vitro mature oocytes from ovarian tissue to preserve women's fertility is still a challenge. At present, there is a therapeutic deadlock for girls and women who need emergency fertility preservation in case of a high risk of ovary invasion by malignant cells. In such a case, ovarian tissue cannot be engrafted; an alternative could be in vitro folliculogenesis. METHODS: This review focuses on the progress of in vitro folliculogenesis in humans. PubMed and Embase databases were used to search for original English-language articles. RESULTS: The first phase of in vitro folliculogenesis is carried out in the original ovarian tissue. The addition of one (or more) initiation activator(s) is not essential but allows better yields and the use of a 3D culture system at this stage provides no added value. The second stage requires a mechanical and/or enzymatic isolation of the secondary follicles. The use of an activator and/or a 3D culture system is then necessary. CONCLUSION: The current results are promising but there is still a long way to go. Obtaining live births in large animals is an essential step in validating this in vitro folliculogenesis technique.

Psychological effect of COVID-19 pandemic among women undergoing infertility care, a French cohort - PsyCovART Psychological effect of COVID-19: PsyCovART.

PURPOSE: To assess psychological state of women who experienced postponement of ART care during the first COVID-19 wave in a French public ward of reproductive medicine. METHODS: An online anonymous survey was emailed between July and August 2020 to all women whose infertility care, including the first consultation for infertility, have been delayed at the beginning of the COVID-19 pandemic. Anxiety, depression, and stress were assessed using Hospital Anxiety and Depression Scale (HADS) and Perceived Stress Scale (PSS-10). Feelings about COVID-19 outbreak, lockdown and suspension of fertility care were assessed by Multiple-Choice Questions and Visual Analog Scales. RESULTS: 435 women answered to the survey (response rate 34.6%). Mean levels of the HADS-A (anxiety), HADS-D (depression) and PSS10 were respectively 7.58(±3.85), 4.51(±3.48), and 27(±6.75). Prevalence of stress was 50.8% and almost half of women presented clear or suggestive anxiety symptoms (respectively 21.6% and 25.7%). Stress and anxiety rates were much higher than those expected in infertile population. Increased stress was observed in women above 35 years and those stopped 'in cycle' or during pre-treatment for in-vitro fertilization or frozen embryo transfer. Patient with history of depression or anxiety had a higher prevalence of perceived stress (p = 0.0006). Postponement was perceived as 'unbearable' for women experiencing stress (p = 0.0032). After the first wave of pandemic, pregnancy desire remained the same and 84.3% of women wanted to resume fertility care as soon as possible. CONCLUSION: Stopping fertility care during the COVID-19 pandemic had a significant psychological impact on women with an increase of stress, and anxiety. Psychological counseling should always be offered especially during this difficult period.

Human ovarian cryopreservation: vitrification versus slow freezing from histology to gene expression.

Cryopreservation of ovarian tissue is one of the strategies offered to girls and women needing gonadotoxic treatment to preserve their fertility. The reference method to cryopreserve is slow freezing; vitrification is an alternative method. The aim was to evaluate which of the two is the best method for human ovarian tissue cryopreservation. Each ovary was divided into three groups: (i) fresh; (ii) slow freezing; and (iii) vitrification. An evaluation of the follicular density, quality and the expression six genes (CYP11A, STAR, GDF9, ZP3, CDK2, CDKN1A) were performed. We observed no significant difference in follicular density within these three groups. Slow freezing altered the primordial follicles compared to the fresh tissue (31.8% vs 55.9%, p = 0.046). The expression of genes involved in steroidogenesis varied after cryopreservation compared to the fresh group; CYP11A was under-expressed in slow freezing group (p = 0.01), STAR was under-expressed in the vitrification group (p = 0.01). Regarding the expression of genes involved in cell cycle regulation, CDKN1A was significantly under-expressed in both freezing groups (slow freezing: p = 0.0008; vitrification: p = 0.03). Vitrification had no effect on the histological quality of the follicles at any stage of development compared to fresh tissue. There was no significant difference in gene expression between the two techniques.

Fertility in men with Klinefelter's syndrome.

Patients with a Klinefelter syndrome (KS), defined by a 47 XXY karyotype, were long considered infertile. Testicular sperm extraction (TESE) now allows them to access fatherhood. We will present the data of studies since first experiment of TESE. Several factors influencing TESE outcome were proposed in these different studies. Among them, clinical and hormonal parameters have reported by few studies, age has been one of the most discussed prognostic factor of positive sperm retrieval rate. Data seems to show that TESE carried out before an age greater than 30 has a poorer prognosis for positive sperm retrieval. In few studies performed in younger patient, before 20 years, SRR was closed to result for 20 to 30 year old patients. Offering a TESE before 16 years old does not improve positive sperm extraction rate. In fact, the few studies carried out before the age of 16 were of poorer prognosis, most often linked to insufficient maturation of the residual gametes. In addition, androgen therapy, frequently prescribed in case of Klinefelter syndrome, did not seem to show any effect on sperm retrieval but only few studies were interested in the possible impact of this treatment. In conclusion, further studies are necessary to determine the interest of new markers to predict the chance of sperm retrieval, taking into account age, hormonal therapy.

Repeated ovarian stimulation with corifollitropin alfa in patients in a GnRH antagonist protocol: no concern for immunogenicity.

BACKGROUND: One injection of corifollitropin alfa replaces the first seven daily FSH injections in controlled ovarian stimulation (COS) cycles. Repeated treatment with therapeutic proteins may cause immune responses or hypersensitivity reactions. We assessed the immunogenicity and safety of corifollitropin alfa treatment in up to three COS cycles. METHODS: In this multicentre, phase III uncontrolled trial, patients (>60 kg) started treatment with one injection of 150 µg corifollitropin alfa on cycle Day 2 or 3 of menses and 0.25 mg ganielix on stimulation Day 5 or 6. Primary outcome measures were antibody formation against corifollitropin alfa (using highly sensitive radioimmunoprecipitation assay), hypersensitivity reactions, local tolerance and adverse events (AEs). RESULTS: First, second and third COS cycles were started by 682, 375 and 198 patients, respectively. No clinically relevant immunogenicity or drug-related hypersensitivity was observed. For 192 patients undergoing their third cycle a post-treatment blood sample was negative in the anti-corifollitropin antibody assay, resulting in an upper limit of the one-sided 95% confidence interval (CI) of 1.5%. Most frequent AEs were procedural pain (17.7%, 95% CI: 14.9-20.8%), headache (9.1%, 95% CI: 7.0-11.5%) and pelvic pain (7.6%, 95% CI: 5.7-9.9%). Cumulative ongoing pregnancy rate after three cycles, including frozen-thawed embryo transfer cycles and spontaneous pregnancies, was 61% (95% CI: 56-65%) after censoring for patients who discontinued. CONCLUSIONS: Treatment with corifollitropin alfa can safely and effectively initiate and sustain ovarian stimulation during the first 7 days of COS in normal responder patients undergoing up to three treatment cycles, without concerns of immunogenicity. The trial was registered under ClinicalTrials.gov identifier NCT00696878.

Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe.

BACKGROUND: The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. METHODS: Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests. RESULTS: Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r=0.82, p<0.05) or calcein AM/ethidium homodimer-1 staining (r=0.76, p<0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60+/-1.1% vs 52.2+/-7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26±8.2% vs 38±4.5%). CONCLUSION: We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

Results of in vitro fertilization versus intrauterine insemination in patients with low anti-Müllerian hormone levels. A single-center retrospective study of 639 + 119 cycles.

OBJECTIVES: The aim of this study was to evaluate the results of in vitro fertilization (IVF) and intrauterine insemination (IUI) in a population of infertile women with low AMH levels, in whom both techniques were possible. METHODS: This was a retrospective analysis of 462 patients treated over 24 months in a single center comparing the live birth rates after 176 IUI and 639 IVF attempts in infertile couples. The women had AMH levels ≤ 1.2 ng/mL and at least one patent tube and their partner's sperm was of sufficient quality for IUI. RESULTS: The live birth rate after IVF was not sufficiently higher than after IUI, or than after IVF attempts converted to IUI for low response (odds ratios in multivariate analysis with respect to IVF: 0.61, p = 0.15 for IUI and 0.73, p = 0.6 for conversions). The pregnancy rates after IVF (13.0 %) and IUI (13.3 %) were similar (p = 0.4), and were non-significantly higher than the pregnancy rate in the IUI conversion group (8.8 %, p = 0.9). Nearly half (43.8 %) of all IVF cycles did not lead to embryo transfer. CONCLUSION: In this group of women with AMH levels ≤ 1.2 ng/mL, IVF did not lead to a higher live birth rate than IUI, and more than 40 % of all IVF attempts did not lead to embryo transfer, suggesting that diminished ovarian reserve is not an indication for IVF over IUI.

A machine learning system with reinforcement capacity for predicting the fate of an ART embryo.

The aim of this work was to construct a score issued from a machine learning system with self-improvement capacity able to predict the fate of an ART embryo incubated in a time lapse monitoring (TLM) system. A retrospective study was performed. For the training data group, 110 couples were included and, 891 embryos were cultured. For the global setting data group, 201 couples were included, and 1186 embryos were cultured. No image analysis was used; morphokinetic parameters from the first three days of embryo culture were used to perform a logistic regression between the cell number and time. A score named DynScore was constructed, the prediction power of the DynScore on blastocyst formation and the baby delivery were tested via the area under the curve (AUC) obtained from the receiver operating characteristic (ROC). In the training data group, the DynScore allowed the blastocyst formation prediction (AUC = 0.634, p < 0.001), this approach was the higher among the set of the tested scores. Similar results were found with the global setting data group (AUC = 0.638, p < 0.001) and it was possible to increase the AUC of the DynScore with a regular update of the prediction system by reinforcement, with an AUC able to reach a value above 0.9. As only the best blastocysts were transferred, none of the tested scores was able to predict delivery. In conclusion, the DynScore seems to be able to predict the fate of an embryo. The reinforcement of the prediction system allows maintaining the predictive capacity of DynScore irrespective of the various events that may occur during the ART process. The DynScore could be implemented in any TLM system and adapted by itself to the data of any ART center.Abbreviations: ART: assisted reproduction technology; TLM: time lapse monitoring system; AUC: area under the curve; ROC: receiver operating characteristic; eSET: elective single embryo transfer; AIS: artificial intelligence system; KID: known implantation data; AMH: anti-Müllerian hormone; BMI: body mass index; WHO: World Health Organization; c-IVF: conventional in-vitro fertilization; ICSI: intracytoplasmic sperm injection; PNf: pronuclear formation; D3: day 3; D5: day 5; D6: day 6; GnRH: gonadotrophin releasing hormone; FSH: follicle stimulating hormone; LH: luteinizing hormone; hCG: human chorionic gonadotropin; PVP: polyvinyl pyrrolidone; PNf: time of pronuclear fading; tx: time of cleavage to x blastomeres embryo; ICM: inner cell mass; TE: trophectoderm; NbCellt: number of cells at t time; FIFO: first in first out; TD: training data group; SD: setting data group; R: real world.

Anti-müllerian hormone levels and antral follicle count in women with a BRCA1 or BRCA2 germline pathogenic variant: A retrospective cohort study.

BACKGROUND: Some studies suggested a decreased ovarian reserve among BRCA1/2 pathogenic variant carriers, with conflicting results. METHODS: We conducted a retrospective single-center observational study of ovarian reserve and spontaneous fertility comparing BRCA1/2 pathogenic variant carriers to controls (women who attended consultations to discuss fertility preservation before gonadotoxic treatment). Measures of associations between plasma AMH concentration, AFC and BRCA1/2 status were modelled by nonlinear generalized additive regression models and logistic regressions adjusted for age at plasma storage, oral contraceptive use, body mass index, cigarette smoking, and the AMH assay technique. RESULTS: The whole population comprised 119 BRCA1/2 pathogenic variant carriers and 92 controls. A total of 110 women (42 carriers, among whom 30 were cancer-free, and 68 controls) underwent an ovarian reserve evaluation. Spontaneous fertility analysis included all women who previously attempted to become pregnant (134 women). We observed a tendency towards a premature decrease in ovarian reserve in BRCA1/2 pathogenic variant carriers, but no difference in mean AMH or AFC levels was found between BRCA1/2 pathogenic variant carriers and controls. An analysis of the extreme levels of AMH (≤5 pmol/l) and AFC (≤7 follicles) by logistic regression suggested a higher risk of low ovarian reserve among BRCA1/2 pathogenic variant carriers (adjusted odds ratio (OR) = 3.57, 95% CI = 1.00-12.8, p = 0.05; and adjusted OR = 4.99, 95% CI = 1.10-22.62, p = 0.04, respectively). DISCUSSION: Attention should be paid to BRCA1/2 pathogenic variant carriers' ovarian reserve, considering this potential risk of premature alteration.

Cellular and Molecular Impact of Vitrification Versus Slow Freezing on Ovarian Tissue.

Objective: To evaluate a vitrification protocol from histology to gene expression to slow freezing. Methods: Ovaries from 12 prepubertal ewes. The same ovary was cut into fragments, studied fresh, frozen, and vitrified. Follicle morphology by hematoxylin-eosin-safran staining, vitality by Trypan Blue, and apoptosis by marking cleaved caspase-3 were studied. The expression of gene: anti-Müllerian hormone (AMH), cytochrome p450 family 11 subfamily A member 1 (CYP11A), and steroidogenic acute regulatory protein (STAR; granulosa cells); growth differentiation factor 9 (GDF9) and zona pellucida glycoprotein 3 (ZP3; oocytes); and cyclin D2 (CCND2) and cyclin-dependent kinase inhibitor 1A (CDKN1A; cell cycle regulation), was evaluated by reverse transcription quantitative polymerase chain reaction. Results: The slow freezing protocol had a significant negative impact on intact primordial follicles compared with fresh tissue (37.6% vs. 62.5%, p = 0.003). More intact follicles after vitrification were observed compared with slow freezing (p = 0.037). The apoptotic primordial follicles were similar after slow freezing and vitrification (12.6% vs. 13.9%). Concerning granulosa cell genes, slow freezing led to a trend toward overexpression of AMH messenger RNA (mRNA; p = 0.07); while vitrification led to a significant overexpression of CYP11A mRNA (p = 0.003), and a trend toward an overexpression of STAR mRNA (p = 0.06). Concerning oocyte genes, both techniques did not lead to a difference of GDF9 and ZP3 mRNA. Concerning cell cycle genes, slow freezing led to a significant underexpression of CCND2 (p = 0.04); while vitrification did not lead to a difference for CCND2 and CDKN1A mRNA. Conclusion: Vitrification preserved follicular morphology better than slow freezing and led to gene overexpressed, while slow freezing led to gene underexpressed. Impact statement The preservation of female fertility and in particular the cryopreservation of ovarian tissue (OT) is a major public health issue aimed at improving the quality of life of patients after gonadotoxic treatments. The use of slow freezing of this OT, which is the reference technique, is not optimal due to tissue alteration. The alternative would be vitrification. This study compares these two techniques. We have highlighted that vitrification preserved follicular morphology better than slow freezing and led to gene overexpressed, while slow freezing led to gene underexpressed.

Simultaneous bilateral tubal ectopic pregnancy after intracytoplasmic sperm injection and embryo transfer, in a patient with Stage 3 endometriosis.

INTRODUCTION: The incidence of extrauterine pregnancy increases to 2-12% following in vitro fertilization -embryo transfer. Several pathogenic theories have been suggested, including abnormal hormonal secretion or exogenous hormones administered in assisted reproductive technology (ART). CASE REPORT: A 32-year-oId nulliparous woman with primary infertility and Stage 3 endometriosis was treated by ART with intracytoplasmic sperm injection and embryo transfer. The patient showed simultaneous bilateral extrauterine pregnancy, managed by laparoscopic salpingectomy. DISCUSSION: The various possible pathophysiological mechanisms are described, with a review of the literature on simultaneous bilateral extrauterine pregnancy following ART. In pregnancies following ART, ectopic pregnancy should always be screened for by serum ß-human chorionic gonadotropin monitoring and transvaginal ultrasound until the implantation site can be confirmed as the incidence is higher than in spontaneous pregnancy. Even if serum ß-human chorionic gonadotropin concentration increases normally, possible bilateral ectopic pregnancy should always be investigated if no intrauterine gestational sac can be seen.