Physiological and psychological stress responses to labor and delivery during COVID-19 pandemic: a cohort study.

OBJECTIVE: To evaluate objective (saliva cortisol) and subjective (questionnaire) stress levels during the Coronavirus disease (COVID-19) pandemic compared to before the pandemic and their effects on obstetric and neonatal outcomes. METHODS: This cohort study included 36 women with low-risk, singleton, term deliveries at a tertiary academic center during the COVID-19 pandemic and 49 who delivered before. Physiological stress was evaluated with salivary cortisol measurements, and emotional stress with stress scale questionnaires (0-10) during active and full dilation stages of labor, and 2-min postpartum. Cord blood cortisol and pH were obtained. Delivery mode, complications, and neonatal outcomes were evaluated. RESULTS: Psychological stress was higher for the COVID-19 group compared to controls during full dilation (6.2 ± 3.4 vs. 4.2 ± 3, p = .009). The COVID-19 group had significantly lower cord cortisol levels (7.3 vs. 13.6 mcg/dl, p = .001). No differences were found regarding salivary cortisol level assessments at active, full dilation and 2-min post-delivery (p = .584, p = .254, p = .829, respectively). No differences were found regarding pH < 7.1 (p = .487), 1- and 5-min Apgar scores < 7 (p = .179) and neonatal weight (p = .958). CONCLUSIONS: Women who delivered during COVID-19 pandemic had higher stress levels at full dilation and lower cord cortisol levels, as may be expected after exposure to a chronic stressor.

Premature ovarian aging in BRCA carriers: a prototype of systemic precocious aging?

PURPOSE: Though former evidence implies a correlation of breast cancer susceptibility gene (BRCA) mutation with reduced ovarian reserve, the data is yet inconsistent. Our aim was to investigate biomarkers of ovarian aging in a cohort of young healthy carriers of the BRCA mutation. We hypothesized that the role played by BRCA genes in aging pathways is not exclusive to the ovary. EXPERIMENTAL DESIGN: Healthy female BRCA carriers, 40 years or younger and healthy male BRCA carriers, 50 years or younger, were enrolled in the study. Serum anti-mullerian Hormone (AMH), fibroblast growth factor-23 (FGF-23), Klotho and IL-1 were measured by enzyme-linked immunosorbent assay (ELISA). Ovarian AMH and protein kinase B (AKT) mRNA from BRCA carriers who underwent prophylactic oophorectomy and from age-matched, healthy, non-carriers who underwent partial oophorectomy due to benign conditions were analyzed by qPCR. RESULTS: Thirty-three female (median age 35y) and 20 male (44y) BRCA carriers were enrolled into the study and matched to control non-carriers (34y and 43y, respectively). Serum AMH level was significantly lower in BRCA female carriers than in both non-carrier controls and age-matched nomograms. The levels of ovarian AMH and AKT mRNA were significantly lower in carriers than in controls. The systemic aging cytokines FGF-23, klotho and IL-1 displayed a differential expression in carriers of both genders. FGF-23 level was higher in carriers (P=0.06). CONCLUSIONS: Our results suggest a link between BRCA mutation, accelerated ovarian aging and systemic aging-related pathophysiology.

Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies.

Anti-Müllerian Hormone Is a Marker for Chemotherapy-Induced Testicular Toxicity.

Due to increased numbers of young cancer patients and improved survival, the impact of anticancer treatments on fertility has become a major health concern. Despite mounting research on ovarian toxicity, there is paucity of data regarding reliable biomarkers of testicular toxicity. Our aim was to evaluate anti-Müllerian hormone (AMH) as a marker for chemotherapy-induced testicular toxicity. Serum AMH and a panel of gonadal hormones were measured in male cancer patients at baseline and after chemotherapy. In the preclinical setting, mice were injected with diverse chemotherapies and were killed 1 week or 1, 3, or 6 months later. We evaluated spermatogenesis by AMH as well as qualitative and quantitative sperm parameters. Nineteen patients were enrolled, the median age was 38 years (21-44 y). Serum AMH was correlated with increased FSH and T and decreased inhibin-B in gonadotoxic protocols (cisplatin or busulfan) and remained unchanged in nongonadotoxic protocols (capecitabine). AMH expression had the same pattern in mice serum and testes; it was negatively correlated with testicular/epididymal weight and sperm motility. The increase in testicular AMH expression was also correlated with elevated apoptosis (terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling) and reduced proliferation (Ki67, proliferating cell nuclear antigen; all seminiferous tubules cells were analyzed). Severely damaged mice testes demonstrated a marked costaining of AMH and GATA-4, a Sertoli cell marker; staining that resembled the pattern of the Sertoli cell-only condition. Our study indicates that the pattern of serum AMH expression, in combination with other hormones, can delineate testicular damage, as determined in both experimental settings. Future large-scale clinical studies are warranted to further define the role of AMH as a biomarker for testicular toxicity.

Long-Term Follow-Up of Chemotherapy-Induced Ovarian Failure in Young Breast Cancer Patients: The Role of Vascular Toxicity.

BACKGROUND: We previously reported that chemotherapy-induced ovarian toxicity may result from acute vascular insult, demonstrated by decreased ovarian blood flow and diminished post-treatment anti-Müllerian hormone (AMH) levels. In the present study, we report the continuous prospective evaluation of ovarian function in that cohort. METHODS: Patients (aged <43 years) with localized breast cancer were evaluated by transvaginal ultrasound prior to initiation of chemotherapy, immediately at treatment completion, and at 6 and 12 months after treatment cessation. Doppler flow velocity indices of the ovarian vasculature (resistance index [RI], pulsatility index [PI]) were visualized. Hormone markers of ovarian reserve were assessed at the same time points. RESULTS: Twenty patients were enrolled in the study. Median age was 34 ± 5.24 years. Ovarian blood flow was significantly reduced immediately following chemotherapy (both RI and PI; p = .01). These parameters were partially recovered at later points of assessment (6 and 12 months after treatment); patients aged <35 years significantly regained ovarian blood flow compared with patients aged >35 years (p < .05). AMH dropped dramatically in all patients following treatment (p < .001) and recovered in only 10 patients. Hormone markers of ovarian reserve shortly after chemotherapy depicted a postmenopausal profile for most patients, accompanied by related symptoms. Follicle-stimulating hormone (FSH) levels recovered in 14 of 20 patients and significantly returned to the premenopausal range in patients aged <35 years (p = .04); 10 of 20 resumed menses at 12 months. The pattern of vascular impairment was lessened in patients treated with a trastuzumab-based protocol, although results did not reach statistical significance (p = .068). CONCLUSION: Continuous prospective evaluation of ovarian vasculature and function in a cohort of young patients during and after chemotherapy indicated that ovarian toxicity may derive from acute vascular insult. Age may affect whether patients regain ovarian function, whereas recovery of blood flow and premenopausal FSH levels at later assessment was notable in patients aged <35 years. IMPLICATIONS FOR PRACTICE: This study explored the role of vascular toxicity in mediating ovarian impairment and recovery following chemotherapy. Continuous prospective evaluation of ovarian vasculature and function in a cohort of young patients during and after chemotherapy indicated that ovarian toxicity may derive from acute vascular insult. Future studies are warranted to further characterize patterns of vascular toxicity of various chemotherapies in clinical practice and to assess the role of chemotherapy-induced vascular toxicity for specific end organs such as the ovary with systemic vascular effect. Elucidating the cause of impairment may facilitate development of measures to minimize vascular toxicity and consequences of acute vascular insult.

A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation.

Granulosa cells support the developing oocytes and serve as transducers of the ovulatory stimulus induced by LH surge. Fyn kinase is expressed in granulosa cells, though its role in these cells has not been studied. In human embryonic kidney 293T cells, microRNA (miR)-125a-3p down-regulates Fyn expression, causing a decrease in cells' migratory ability. Our aim was to explore the role of miR-125a-3p and Fyn in granulosa cells toward ovulation, focusing on migration as a possible mechanism. We demonstrate expression of miR-125a-3p and Fyn in mouse mural granulosa cells of preovulatory follicles and miR-125a-3p-induced down-regulation of Fyn expression in a granulosa cell line (rat). Administration of human chorionic gonadotropin (hCG; LH analog) caused a 75% decrease in the in vivo miR-125a-3p:Fyn mRNA ratio, followed by a 2-fold increased migratory ability of mural granulosa cells. In the hCG-treated granulosa cell line, miR-125a-3p expression was decreased, followed by Fyn up-regulation and phosphorylation of focal adhesion kinase and paxillin, enabling cell migration. An in vivo interference with miR-125a-3p:Fyn mRNA ratio in granulosa cells by intrabursal injections of Fyn small interfering RNA or miR-125a-3p mimic caused a 33 or 55% decrease in the number of ovulated oocytes, respectively. These observations reveal a new regulatory pathway in mural granulosa cells under the regulation of LH/hCG. Modulation of cell migration may account for the significance of the LH/hCG-miR-125a-3p-Fyn pathway to ovulation.

Gonadotrophin-releasing hormone agonists for fertility preservation: unraveling the enigma?

STUDY QUESTION: Can gonadotrophin-releasing hormone agonists (GnRH-a) preserve long-term fertility when administered prior to and concomitantly with chemotherapy? SUMMARY ANSWER: GnRH-a display a differential protective effect on fertility, depending upon the specific chemotherapy-induced mechanism of ovarian injury. WHAT IS KNOWN ALREADY: The role of GnRH-a in fertility preservation has been constantly debated and their use is considered experimental due to conflicting clinical evidence and paucity of data regarding their mechanism for ovarian protection. STUDY DESIGN, SIZE, DURATION: In vivo model: 7-8 weeks old imprinting control region (ICR) mice were injected with GnRH-a (Leuprolide-acetate) or saline prior to and concomitantly with cyclophosphamide, doxorubicin or saline and sacrificed at various time-points on a longitudinal follow-up; 24 h (n = 36), 1 week (n = 40), 1 month (n = 36) and 9 months (n = 66) post chemotherapy treatment. Blood samples were drawn on Day 0 and on a monthly basis after chemotherapy treatment. On the day of sacrifice, blood samples were drawn and ovaries excised and processed for either immunohistochemistry (IHC), protein or RNA extraction. In vitro model: 21-23 days old Wistar-derived rats were sacrificed, their ovaries excised and primary granulosa cells (PGC) were either isolated for in vitro culture, or processed for immunofluorescence (IF) as well as for protein or RNA extraction. MATERIALS, SETTING, METHODS: Ovarian reserve was estimated by serial measurements of serum anti-mullerian hormone (AMH), quantified by the AMH Gen II ELISA assay. Ovarian AMH and phosphorylated Akt (pAkt) were detected by immunoblotting. Vascular endothelial growth factor (VEGF) was measured by quantitative PCR. Ovarian GnRH receptor (GnRHR), AMH and CD34 were visualized by IHC, and apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL). MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide-induced ovarian injury caused a prompt decrease in AMH level (P < 0.01) and a further long-term decline in serum AMH (P = 0.017), indicating damage to the ovarian reserve. Pretreatment with GnRH-a diminished AMH-decrease (P < 0.05) and maintained serum AMH level in the long run (P < 0.05). Doxorubicin-exerted ovarian-vascular-injury is also displayed by an acute increase in ovarian VEGF level (P < 0.05) and a sustained decrease in serum AMH level (P < 0.001). This was followed by ovarian recovery manifested by increased neovascularization. GnRH-a delayed the recovery in AMH level and decreased the level of VEGF (P < 0.001), thus interfering with the vascular recovery subsequent to doxorubicin-induced vascular damage. LIMITATIONS, REASONS FOR CAUTION: To portray the differential mechanism of each chemotherapy, cyclophosphamide and doxorubicin were given separately, whereas most of the clinical protocols include several types of chemotherapies. Thus, future study should explore a prospective evaluation of various chemotherapies, as well as combined chemotherapeutic protocols. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that different chemotherapy agents affect the ovary via diverse mechanisms and thus the administration of GnRH-a concomitantly, could be beneficial to a subpopulation of patients treated with cyclophosphamide-based protocols. STUDY FUNDING/COMPETING INTERESTS: This work was partially supported by a grant from the Israel Science Foundation (ISF) to I.B.-A. The authors have no conflict of interest to disclose.

Interleukin-1 deficiency prolongs ovarian lifespan in mice.

Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1ß-KO mice. IL-1α-KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α-KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α-KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1ß-KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α-KO mice. The protein and mRNA of both IL-1α and IL-1ß mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2-associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1ß, IL-6, and TNF-α in ovaries of IL-1α-KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways.

Hormonal regulation of pigment epithelium-derived factor (PEDF) expression in the endometrium.

Pigment epithelium-derived factor (PEDF) is highly expressed in the female reproductive system and is subjected to regulation by steroid hormones in the ovary. As the uterine endometrium exhibits morphological and functional changes in response to estrogen (E2) and progesterone (P4), we aimed at characterizing the expression of PEDF in this component of the female reproductive tract and further at exploring the hormonal regulation of its expression. We found that PEDF is expressed in human and mouse endometrium. We further showed that this expression is subjected to regulation by steroid hormones, both in vivo and in vitro, as follows: E2 decreased PEDF expression and P4 increased its levels. In human endometrial samples, PEDF levels were dynamically altered along the menstrual cycle; they were low at the proliferative and early secretory phases and significantly higher at the late secretory phase. The expression levels of PEDF were inversely correlated to that of vascular endothelial growth factor (VEGF). We also showed that PEDF receptor was expressed in the endometrium and that its stimulation reduced VEGF expression. Illustrating the pattern of PEDF expression during the menstrual cycle may contribute to our understanding of the endometrial complexity.

A physiological approach for treating endometriosis by recombinant pigment epithelium-derived factor (PEDF).

STUDY QUESTION: Is pigment epithelium-derived factor (PEDF) expressed in the rodent endometrium and can it be utilized to treat endometriosis without negatively affecting reproductive parameters? SUMMARY ANSWER: PEDF is dynamically expressed in rat endometrium throughout the estrous cycle in a reciprocal manner to vascular endothelial growth factor (VEGF); it possesses potent therapeutic properties for endometriosis that do not compromise the reproductive parameters. WHAT IS KNOWN ALREADY: Endometriosis pathogenesis depends mainly on neovascularization, with a high local level of VEGF. PEDF, a 50 kDa secreted glycoprotein with a potent anti-angiogenic activity, negates several strong pro-angiogenic factors, such as VEGF. STUDY DESIGN, SIZE, DURATION: Rat endometrial samples were collected at various days of the estrous cycle (n = 5 rats/day) and mRNA of VEGF and PEDF was determined. Endometriosis was induced by transplanting uterine pieces onto the inner surface of the abdominal wall of recipient rats, resulting in proliferation of the endometrial transplants. Recipient rats were randomly injected intravenously (IV), every third day for the next 3 weeks, with either Tris ('control'; n = 7) or recombinant PEDF (rPEDF; 2 mg/kg/day; 'PEDF prevention'; n = 7), while others were IV injected every third day starting from Day 9 after grafting until the end of 3 weeks, with rPEDF (2 mg/kg/day; 'PEDF treatment'; n = 6). The effect of rPEDF on the duration of the estrous cycle and on the number of ovulated oocytes was evaluated in rats that were randomly divided into four groups and were injected with either Tris or rPEDF every third day for 3 weeks: naive rats (n = 6); rPEDF-treated rats (n = 5); endometriosis-induced rats (n = 5); or endometriosis + rPEDF rats (n = 6). MATERIALS, SETTING, METHODS: Reproductive parameters: the estrous cycle was evaluated by daily vaginal smears, and the number of ovulated oocytes in the oviductal ampullae of estrus rats was counted. The efficiency of endometriosis induction and treatment was evaluated on the third week after endometrial transplantation, on the day of pro-estrus. Endometrial transplants were isolated and weighted. PEDF and VEGF were monitored by quantitative PCR and immunohistochemistry using confocal microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: PEDF mRNA and protein were dynamically expressed in the endometrium all throughout the estrous cycle, reciprocally to VEGF; VEGF was highly expressed during estrus while PEDF expression was low, and vice versa at metestrus II. The weight of the endometrial transplants was significantly reduced after PEDF administration (13% of control for 'PEDF treatment' rats; 7% of control for 'PEDF prevention' rats; P < 0.001). Histology of the transplants' remnants showed a complete loss of their endometrial characteristics. Furthermore, the level of VEGF mRNA in the transplants of PEDF-administered rats was significantly lower (P < 0.05) than in transplants of control rats. Administration of rPEDF had no effect on the estrous cycle or ovulation rate of naive rats, while it had a significantly beneficial effect on the low ovulation rate of endometriosis-induced rats (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The experiments were performed in a rat model. WIDER IMPLICATIONS OF THE FINDINGS: The endometriosis therapeutic potency of PEDF that is exerted reciprocally to VEGF and does not compromise reproductive parameters offers a rational for using PEDF as a treatment for endometriosis with a potential of treating other reproductive angiogenic-related pathologies.

Chemotherapy-induced ovarian failure as a prototype for acute vascular toxicity.

BACKGROUND: Chemotherapy-related amenorrhea is a frequent side effect observed in young breast cancer patients. Studies in mice revealed that chemotherapy-induced gonadal toxicity may result from vascular damage. We prospectively evaluated ovarian blood flow and function in young breast cancer patients following chemotherapy. METHODS: Young female patients with localized breast cancer undergoing adjuvant or neoadjuvant anthracycline- or taxane-based chemotherapy were evaluated using transvaginal ultrasound prior to initiation of and immediately after cessation of chemotherapy. Doppler-flow velocity indices of the ovarian vasculature-resistance index (RI), pulsatility index (PI)-and size measurements were visualized. Hormonal profiles, anti-Müllerian hormone (AMH) levels, and menopausal symptoms were assessed at the same time points. RESULTS: Twenty breast cancer patients were enrolled in the study. The median age was 34 ± 5.24 years. Ovarian blood flow was significantly reduced shortly following chemotherapy: RI decreased by 52.5% and PI decreased by 24.2%. The mean ovarian size declined by 19.08%. Patients who were treated with sequential chemotherapy experienced further reductions in ovarian blood flow and ovarian size after the second sequence. AMH levels dropped dramatically in all patients following treatment. Hormonal profiles after treatment depicted a postmenopausal profile for most patients, accompanied by related symptoms. CONCLUSIONS: Our results may imply a mechanism of chemotherapy-induced ovarian toxicity manifested by decreased ovarian blood flow accompanied by a reduction in ovarian size and diminished post-treatment AMH levels. Based upon our former preclinical studies, we assume that this may derive from an acute insult to the ovarian vasculature and may represent an initial event triggering a generalized phenomenon of end-organ toxicity.