In Vitro Maturation of Oocytes Obtained from Ovarian Cortex Among Postpubertal Hematological Cancer Patients Undergoing Fertility Preservation.

Purpose: In vitro maturation (IVM) of oocytes obtained from ovarian tissue during ovarian tissue cryopreservation (OTC) is a technique for fertility preservation in patients with cancer obviating the need to postpone chemotherapy initiation. Little is known about IVM outcomes in hematological malignancies, especially post-chemotherapy. The purpose of this study was to evaluate the effect of cytotoxic treatment on the potential to retrieve immature oocytes and mature them in vitro and examine the association between serum inflammatory markers and these results. Methods: In this retrospective study, we evaluated inflammation markers, including B symptoms and IVM outcomes of 78 chemotherapy-naive and exposed patients diagnosed with Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML). Results: The mean number of oocytes found was 7.2 ± 7.2. The average number of oocytes matured by IVM was 2.8 ± 3.5, and a mean IVM rate was 32.1 ± 27.7%. All patients in the ALL and AML groups had previous exposure to chemotherapy before OTC, compared with 50.0% (7/14) and 31.9% (15/47) in the NHL and HL groups, respectively. Among patients with lymphoma, chemotherapy exposure was associated with the reduced number of retrieved oocytes (9.8 ± 7.7 vs. 5.3 ± 5.7 oocytes, p = 0.049) in the HL group but not with the number of mature oocytes or IVM rate. B symptoms were not associated with IVM outcomes. Lymphocyte count (ß = 1.584; p = 0.038) and lactate dehydrogenase (ß = 0.009; p = 0.043) were the only significant parameters associated with the number of matured oocytes in a linear regression model. Conclusion: IVM is a promising assisted reproductive technology, which holds great potential for patients in need of urgent fertility preservation or those who cannot receive hormonal stimulation. Our results demonstrate the feasibility of the technique even in the presence of B symptoms and elevated inflammation markers and in patients with previous exposure to chemotherapy.

Changes in Sperm Parameters with Time in Men with Normal and Abnormal Baseline Semen Analysis.

The association between paternal age and sperm quality in the population level has been previously studied. Only limited data exists regarding the intra-personal variations in semen parameters among fertile and infertile men over time. We aimed to assess trends over time in semen parameters among men with normal and abnormal baseline sperm parameters and investigate potential risk factors for sperm quality deterioration. This retrospective cohort study was conducted at a university-affiliated medical center in vitro fertilization (IVF) unit. Patients with at least two semen analyses (SA) performed > 1 year apart, with the last SA done between 2017 and 2021, were included. The study consisted of two main analyses-comparison of intra-patient's sperm parameters changes in men with normal and abnormal baseline SA (BSA) and analysis of risk factors for developing abnormal semen parameters over time in men who had normal BSA parameters. This study included a total of 902 men assessed for infertility with normal and abnormal BSA. The average time interval between tests was 1015 days (range 366-7709 days). Among individuals with normal BSA, there was a mild decline in most parameters-concentration (- 6.53 M/ml), motility (- 7.74%), and total motile count (TMC) (- 21.80 M) (p < 0.05 for all parameters). In contrast, a slight improvement in most parameters, except for concentration, was noted in men with abnormal BSA-volume (+ 0.21 ml), motility (+ 8.72%), and TMC (+ 14.38 M) (p < 0.05 for all parameters). Focusing on men with normal BSA, 33.5% of individuals developed abnormality in one or more of their sperm parameters over time, within a mean time of 1013 ± 661 days. We also found that only time between tests emerged as an independent prognostic factor for the development of abnormal SA later. Interestingly, sperm deterioration in participants in their third, fourth, and fifth decades of life with normal initial semen analysis was similar. Our study provides evidence of a decline in semen quality over time in individuals with normal BSA, in contrast to men with abnormal BSA. Longer time intervals between tests independently increase the risk of sperm abnormalities.

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

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

Dydrogesterone supplementation in addition to routine micronized progesterone administration for luteal support in cycles triggered with lone GnRH agonist results in an acceptable pregnancy rate and avoids the need to freeze embryos.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is reduced when using antagonist cycle with gonadotrophin releasing hormone (GnRH) agonist trigger before ovum pick up. This trigger induces short luteinizing hormone (LH) and follicle stimulating hormone (FSH) peaks, resulting in an inadequate luteal phase and a reduced implantation rate. We assessed whether the luteal phase can be rescued by supplementing with oral dydrogesterone (duphaston) in antagonist cycles after a lone GnRH agonist trigger. METHODS: A retrospective cohort study. The study group (N.=123) included women who underwent IVF. Patients received a GnRH-antagonist with a lone GnRH-agonist trigger due to imminent OHSS. The control group (N.=374) included patients who underwent a standard antagonist protocol with a dual trigger of a GnRH-agonist and human chorionic gonadotrophin (hCG). All the patients were treated with micronized progesterone (utrogestan) for luteal phase support. Study patients were given duphaston in addition. RESULTS: The fertilization rate was comparable between the two groups. The mean number of embryos transferred, the clinical pregnancy rate and the take-home baby rate were comparable between groups (1.5±0.6 vs. 1.5±0.5 and 46.3% vs. 41.2%, and 66.7% vs. 87.7%, respectively). No OHSS event was reported in either group. CONCLUSIONS: This study was the first to evaluate outcomes of duphaston supplementation for luteal support in an antagonist cycle with lone GnRH agonist trigger. The functionality of the luteal phase of those cycles could be restored by adding duphaston. This approach was found to be safe and prevented the need to postpone embryo transfer in case of pending OHSS.

Sperm quality is not affected by the BNT162b2 mRNA SARS-CoV-2 vaccine: results of a 6-14 months follow-up.

PURPOSE: We aimed to investigate the possible effect of SARS-CoV-2 vaccination on sperm quality by evaluating semen analyses of men prior to vaccination and 6-14 months after vaccination. METHODS: This was a retrospective cohort study, conducted in a university-affiliated in vitro fertilization center between October 2021 and March 2022, including men not previously infected with the SARS-CoV-2 virus who received at least 2 doses of the Pfizer-BioNTech (BNT162b2) SARS-CoV-2 vaccine. Semen analyses of samples given pre-vaccination and 6-14 months post-vaccination were analyzed for the parameters of volume, concentration, motility, morphology, and total motile count (TMC) and compared. These parameters were also compared separately for men who received a third (booster) dose and for men with pre-vaccination normal and abnormal sperm. Correlations between time from vaccination and post-vaccination sperm parameters were also assessed. RESULTS: Fifty-eight men were included in the final analysis. Semen volume (2.9 ± 1.4 vs. 2.9 ± 1.6 ml), sperm concentration (42.9 ± 37.9 vs. 51.5 ± 46.2 million/ml), motility (42.5 ± 23.1 vs. 44.3 ± 23.4 percent), morphology (8.8 ± .16.6 vs. 6.6 ± 8.8 percent), and TMC (55.7 ± 57.9 vs. 71.1 ± 77.1 million) were comparable between the pre- and post-vaccination samples. This was true for the entire study cohort, for the subgroup of men who received a third dose and for the subgroups of men with a pre-vaccination normal and abnormal semen samples. No correlation was found between time from vaccination and post-vaccination sperm parameters. CONCLUSIONS: The Pfizer-BioNTech (BNT162b2) SARS-CoV-2 vaccine does not impair any of the sperm parameters over a relatively long-time interval of 6 to 14 months from vaccination.

Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues.

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design: Experimental cryopreservation of testicular tissue. Setting: University Medical Center. Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment. Interventions: None. Main Outcome Measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining. Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment. Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration. Clinical Trial Registration Number: NIH research clinical trials number: NCT02529826.

Pre-pubertal oocytes harbor altered histone modifications and chromatin configuration.

Pre-pubertal oocytes are still dormant. They are arrested in a GV state and do not undergo meiotic divisions naturally. A multitude of molecular pathways are changed and triggered upon initiation of puberty. It is not yet clear which epigenetic events occur in oocytes upon pubertal transition, and how significant these epigenetic events may be. We evaluated epigenetic marker levels in mouse pre-pubertal and post-pubertal female oocytes. In addition, we evaluated H3K9me2 levels in human oocytes collected from fertility preservation patients, comparing the levels between pre-pubertal patients and post-pubertal patients. The chromatin structure shows a lower number of chromocenters in mouse post-pubertal oocytes in comparison to pre-pubertal oocytes. All heterochromatin marker levels checked (H3K9me2, H3K27me3, H4K20me1) significantly rise across the pubertal transition. Euchromatin markers vary in their behavior. While H3K4me3 levels rise with the pubertal transition, H3K27Ac levels decrease with the pubertal transition. Treatment with SRT1720 [histone deacetylase (HDAC) activator] or overexpression of heterochromatin factors does not lead to increased heterochromatin in pre-pubertal oocytes. However, treatment of pre-pubertal oocytes with follicle-stimulating hormone (FSH) for 24 h - changes their chromatin structure to a post-pubertal configuration, lowers the number of chromocenters and elevates their histone methylation levels, showing that hormones play a key role in chromatin regulation of pubertal transition. Our work shows that pubertal transition leads to reorganization of oocyte chromatin and elevation of histone methylation levels, thus advancing oocyte developmental phenotype. These results provide the basis for finding conditions for in-vitro maturation of pre-pubertal oocytes, mainly needed to artificially mature oocytes of young cancer survivors for fertility preservation purposes.

Reduced Endothelin-2 and Hypoxic Signaling Pathways in Granulosa-Lutein Cells of PCOS Women.

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210-designated hypoxiamiR-and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.

Age-Dependent in vitro Maturation Efficacy of Human Oocytes - Is There an Optimal Age?

In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over ovarian tissue cryopreservation (OTC), as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that in vitro maturation (IVM) performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. We conducted a retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM. A total of 133 chemotherapy naïve patients aged 1-35 years were included in the study. The primary outcome was IVM rate in the different age groups - pre-menarche (1-5 and ≥6 years), post-menarche (menarche-17 years), young adults (18-24 years) and adults (25-29 and 30-35 years). We demonstrate a gradual increase in mean IVM rate in the age groups from 1 to 25 years [4.6% (1-5 years), 23.8% (6 years to menarche), and 28.4% (menarche to 17 years)], with a peak of 38.3% in the 18-24 years group, followed by a decrease in the 25-29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30-35 years group. A significant difference in IVM rates was noted between the age extremes - the very young (1-5 years) and the oldest (30-35 years) groups, as compared with the 18-24-year group (p < 0.001). Importantly, number of oocytes matured, percent of patients with matured oocytes, and overall maturation rate differed significantly (p < 0.001). Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggests that oocytes retrieved during OTC prior to chemotherapy have an optimal window of age that shows higher success rates, suggesting that oocytes may have an inherent tendency toward better maturation in those age groups.

Chemotherapy-based gonadotoxicity risk evaluation as a predictor of reproductive outcomes in post-pubertal patients following ovarian tissue cryopreservation.

BACKGROUND: The sterilizing effect of cancer treatment depends mostly on the chemotherapy regimen and extent of radiotherapy. Prediction of long-term reproductive outcomes among cancer survivors according to chemo-radiotherapy regimen may improve pre-treatment fertility preservation counseling and future reproductive outcomes. METHODS: The aim of this study was to evaluate long term reproductive outcomes in cancer survivors according to gonadotoxicity risk estimation of the chemo-radiotherapy regimens utilized. This retrospective cohort study was comprised of post-pubertal female patients referred for fertility preservation during 1997 and 2017 was performed. Eligible adult patients were addressed and asked to complete a clinical survey regarding their ovarian function, menstruation, reproductive experience and ovarian tissue auto-transplantation procedures. Results were stratified according to the gonadotoxic potential of chemotherapy and radiotherapy they received-low, moderate and high-risk, defined by the regimen used, the cumulative dose of chemotherapy administered and radiation therapy extent. RESULTS: A total of 120 patients were eligible for the survey. Of those, 92 patients agreed to answer the questionnaire. Data regarding chemotherapy regimen were available for 77 of the 92 patients who answered the questionnaire. Menopause symptoms were much more prevalent in patients undergoing high vs moderate and low-risk chemotherapy protocol. (51.4% vs. 27.3% and 16.7%, respectively; p < 0.05). Spontaneous pregnancy rates were also significantly lower in the high-risk compared with the low-risk gonadotoxicity regimen group (32.0% vs. 58.3% and 87.5%, respectively; p < 0.05). CONCLUSION: Patients scheduled for aggressive cancer treatment have significantly higher rates of menopause symptoms and more than double the risk of struggling to conceive spontaneously. Improving prediction of future reproductive outcomes according to treatment protocol and counseling in early stages of cancer diagnosis and treatment may contribute to a tailored fertility related consultation among cancer survivors.

In vitro maturation rates in young premenarche patients.

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

Antral follicle count recovery in women with menses after treatment with and without gonadotropin-releasing hormone agonist use during chemotherapy for breast cancer.

PURPOSE: After chemotherapy for breast cancer, most women will recover some ovarian function, but the timing and extent of this recovery are poorly understood. We studied post-chemotherapy ovarian recovery in women with and without a history of ovarian suppression during chemotherapy. METHODS: Reproductive age breast cancer patients who were seen prior to chemotherapy for fertility preservation consult were consented for follow-up ovarian function assessment (every 3-6 months after chemotherapy) with antral follicle count (AFC) in this prospective cohort study. We restricted our analysis to those with menses present after chemotherapy. Box plots were used to demonstrate the change in follow-up AFC versus time elapsed after chemotherapy. A mixed effects regression model was used to assess differences in AFC. RESULTS: Eighty-eight patients with a history of newly diagnosed breast cancer were included. Forty-five patients (51%) had ovarian suppression with GnRH agonist (GnRHa) during chemotherapy. AFC recovery appeared to plateau at 1 year after completing chemotherapy at a median of 40% of pre-chemotherapy AFC. After adjustment for age, initial AFC, cyclophosphamide exposure, combined hormonal contraceptive (CHC) use, and tamoxifen use, AFC recovered faster and to a greater degree for those women who underwent GnRHa therapy for ovarian protection during chemotherapy (P = 0.032). CONCLUSIONS: Women with menses after chemotherapy for breast cancer appear to recover their full potential AFC 1 year after their last chemotherapy dose. Treatment with GnRHa during chemotherapy is associated with a higher degree of AFC recovery. The findings of this study can aid in counseling patients prior to chemotherapy about expectations for ovarian recovery and planning post-treatment fertility preservation care to maximize reproductive potential when pre-treatment fertility preservation care is not possible or has limited oocyte yield.

Preimplantation genetic diagnosis as a strategy to prevent having a child born with an heritable eye disease.

BACKGROUND: In developed countries, genetically inherited eye diseases are responsible for a high percentage of childhood visual impairment. We aim to report our experience using preimplantation genetic diagnostics (PGD) in order to avoid transmitting a genetic form of eye disease associated with childhood visual impairment and ocular cancer. MATERIAL AND METHODS: Retrospective case series of women who underwent in vitro fertilization (IVF) and PGD due to a familial history of inherited eye disease and/or ocular cancer, in order to avoid having a child affected with the known familial disease. Each family underwent genetic testing in order to identify the underlying disease-causing mutation. IVF and PGD treatment were performed; unaffected embryos were implanted in their respective mothers. RESULTS: Thirty-five unrelated mothers underwent PGD, and the following hereditary conditions were identified in their families: albinism (10 families); retinitis pigmentosa (7 families); retinoblastoma (4 families); blue cone monochromatism, achromatopsia, and aniridia (2 families each); and Hermansky-Pudlak syndrome, Leber congenital amaurosis, Norrie disease, papillorenal syndrome, primary congenital cataract, congenital glaucoma, Usher syndrome type 1F, and microphthalmia with coloboma (1 family each). Following a total of 88 PGD cycles, 18 healthy (i.e., unaffected) children were born. CONCLUSIONS: Our findings underscore the importance an ophthalmologist plays in informing patients regarding the options now available for using prenatal and preimplantation genetic diagnosis to avoid having a child with a potentially devastating genetic form of eye disease or ocular cancer. This strategy is highly relevant, particularly given the limited options currently available for treating these conditions.

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.

Random start ovarian stimulation for fertility preservation appears unlikely to delay initiation of neoadjuvant chemotherapy for breast cancer.

STUDY QUESTION: Is random start ovarian stimulation associated with delays in initiation of neoadjuvant chemotherapy for breast cancer? SUMMARY ANSWER: Among women who complete fertility preservation (FP) consultation, random start ovarian stimulation is unlikely to delay time to initiation of neoadjuvant chemotherapy start. WHAT IS KNOWN ALREADY: Neoadjuvant chemotherapy is now a widely accepted treatment modality for operable breast cancer and random start ovarian stimulation is an increasingly-utilized modality for FP. While conventional ovarian stimulation does not appear to delay starting adjuvant chemotherapy, the relationship between random start ovarian stimulation and neoadjuvant chemotherapy start is not well-understood. STUDY DESIGN, SIZE, DURATION: Cross-sectional study of all women seen between from January 2011 to April 2017 for FP consultation prior to starting neoadjuvant chemotherapy for breast cancer. PARTICIPANTS/MATERIALS, SETTING, METHODS: A chart-review was performed. Study inclusion criteria were female sex; age 18-45; non-metastatic breast cancer diagnosis; underwent FP consultation; underwent neoadjuvant chemotherapy. Referrals for FP evaluation came from a regional referral base of oncology clinics. Various time-points related to cancer diagnosis, FP or chemotherapy were obtained from medical record review. We compared time-points between those who underwent ovarian stimulation for FP versus those who did not using T-tests and linear modeling. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 89 women who had FP consultation prior to neoadjuvant chemotherapy were identified. Sixty-seven percent underwent ovarian stimulation prior to cancer treatment and 33% did not. Women who underwent ovarian stimulation were similar in parity and clinical cancer stage to those who did not. Overall, the average time from cancer diagnosis to chemotherapy start was similar between the group that did undergo ovarian stimulation and those who did not (38.1 ± 11.3 versus 39.4 ± 18.5 days, P = 0.672). Those that underwent ovarian stimulation were referred 9.4 ± 6.8 days after diagnosis versus 17.9 ± 15.3 days for those who did not undergo ovarian stimulation (P < 0.001). LIMITATIONS REASONS FOR CAUTION: Retrospective study with potential for selection bias among those who underwent ovarian stimulation versus those who did not. Reasons for caution include the possibility of unmeasured differences among those who did and did not undergo ovarian stimulation, including: patients' and providers' perceptions of the urgency to start chemotherapy, ongoing oncology work-up and treatment planning, FP decision-making, and the pursuit of second and third opinions. The difference in time from referral to FP consultation may have also influenced patients' decisions about whether to undergo ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: In this study, FP with random start ovarian stimulation was not associated with a delay cancer treatment in the neoadjuvant setting, so long as there was a prompt FP referral. Patients undergoing neoadjuvant chemotherapy should be informed of these findings to avoid unnecessary anxiety due to concern for delays. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by departmental research funding within the University of California, San Francisco Department of Obstetrics, Gynecology and Reproductive Sciences. There are no conflicts of interest to declare.

Elevated circulating micro-ribonucleic acid (miRNA)-200b and miRNA-429 levels in anovulatory women.

OBJECTIVE: To study the role of micro-RNA (miRNA)-200b and miRNA-429 in human ovulation and to measure their expression levels in ovulatory and anovulatory patients. DESIGN: Micro-RNA-200b and miRNA-429 expression analysis in human serum and granulosa cells at different phases of the ovulation cycle in normal cycling women and women undergoing assisted reproductive technology cycles. SETTING: University-affiliated hospital and academic research laboratory. PATIENT(S): Forty women (7 normally ovulating, 15 normally ovulating with pure male infertility factor, and 18 with polycystic ovary syndrome) were included in this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression profile of circulating miRNAs and granulosa cells was assessed by means of real-time quantitative reverse transcription-polymerase chain reaction analysis. RESULT(S): We identified miRNA-200b and miRNA-429 in the sera of all women tested. These miRNA expression levels were elevated during the early follicular phase of the cycle compared with serum levels during the early luteal phase. Anovulatory women, diagnosed with polycystic ovary syndrome, expressed significantly higher levels of miRNA-200b and miRNA-429 compared with spontaneously ovulating women. Ovulation induction with exogenous gonadotropins during an IVF cycle reduced these levels to the levels measured in normal ovulating women. CONCLUSION(S): Our findings suggest an involvement of miRNA-200b and miRNA-429 in the pituitary regulation of human ovulation. Although it is unclear whether this altered miRNA expression profile is a cause or a result of anovulation, the levels of these molecules in the serum of anovulatory women may serve as serum biomarkers for the ovulation process.

microRNA in Human Reproduction.

microRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They emerged more than 20 years ago as key posttranscriptional regulators of gene expression. The regulatory role of these small RNA molecules has recently begun to be explored in the human reproductive system. microRNAs have been shown to play an important role in control of reproductive functions, especially in the processes of oocyte maturation, folliculogenesis, corpus luteum function, implantation, and early embryonic development. Knockout of Dicer, the cytoplasmic enzyme that cleaves the pre-miRNA to its mature form, results in postimplantation embryonic lethality in several animal models, attributing to these small RNA vital functions in reproduction and development. Another intriguing characteristic of microRNAs is their presence in body fluids in a remarkably stable form that is protected from endogenous RNase activity. In this chapter we will describe the current knowledge on microRNAs, specifically relating to human gonadal cells. We will focus on their role in the ovarian physiologic process and ovulation dysfunction, regulation of spermatogenesis and male fertility, and putative involvement in human normal and aberrant trophoblast differentiation and invasion through the process of placentation.

HIF1A-dependent increase in endothelin 2 levels in granulosa cells: role of hypoxia, LH/cAMP, and reactive oxygen species.

Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 µM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.

Altered endothelin expression in granulosa-lutein cells of women with polycystic ovary syndrome.

AIMS: To examine the levels of endothelin system components in granulosa lutein cells (GLCs) of women with PCOS and compare them to normally ovulating women undergoing In Vitro Fertilization (IVF). Polycystic ovary syndrome (PCOS) is one of the most common endocrine-metabolic disorders in women of reproductive age. Endothelins are locally produced by endothelial and granulosa cells of the preovulatory follicle. Abnormal expression or production of endothelins may be a contributing factor in PCOS pathogenesis. MAIN METHODS: Follicular aspirates containing GLCs were obtained from PCOS and normally ovulating patients undergoing oocyte retrieval during the IVF cycle. RNA was extracted and endothelin system components were quantified using real-time PCR. GLCs were cultured in basal media for 7 days, and then challenged with various luteinizing agents (luteinizing hormone, hCG, or forskolin) for 24 h. KEY FINDINGS: In GLCs from women with PCOS, Endothelin-1 mRNA expression was elevated (2.2-fold) as compared with normally ovulating women, whereas endothelin-2 mRNA was reduced (1.8-fold). ET receptors and endothelin-converting enzyme showed the same expression levels in the two groups. In vitro modeling showed that although the steroidogenic response was preserved in GLC, endothelin expression levels were not exhibited in vitro in their original pattern. SIGNIFICANCE: Dysregulation of ovarian endothelin expression may induce a pathologic ovulation pattern characteristic of PCOS.

Reproductive outcome of fresh or frozen-thawed embryo transfer is similar in high-risk patients for ovarian hyperstimulation syndrome using GnRH agonist for final oocyte maturation and intensive luteal support.

BACKGROUND: Triggering ovulation by GnRH agonist (GnRHa) in GnRH antagonist IVF protocols coupled with adequate luteal phase support has recently been suggested as a means to prevent ovarian hyperstimulation syndrome (OHSS). Our objective was to examine the outcome of fresh embryo transfer (f-ET) after triggering ovulation by GnRHa and providing intensive luteal phase supplementation, compared with that of the next first frozen-thawed embryo transfer (ft-ET) after cycles with the same protocol and cryopreservation of all the embryos. METHODS: We performed a cohort study at a university-based IVF clinic. The study population was patients at high risk for OHSS. A daily dose of 50 mg i.m. progesterone in oil and 6 mg of oral 17-ß-estradiol initiated on oocyte retrieval day in the f-ET group (n= 70). In the ft-ET group (n= 40) the embryos were cryopreserved and transferred in the next cycle. RESULTS: The live birth rate per f-ET was 27.1 versus 20% in the ft-ET groups [P = 0.4; rate ratio = 1.36 (0.65-2.81)]. The implantation, pregnancy and spontaneous abortion rates were comparable in both groups. None of the patients developed OHSS. CONCLUSIONS: In this observational cohort study, we showed that triggering ovulation with GnRHa and intensive luteal phase support is a promising new modality to prevent OHSS without the cost of cycle cancellation, ET deferral and reduced clinical pregnancy rates. Confirmation of these findings by RCTs is now required.