Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

STUDY QUESTION: Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? SUMMARY ANSWER: EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). WHAT IS KNOWN ALREADY: The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. STUDY DESIGN, SIZE, DURATION: This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. STUDY FUNDING/COMPETING INTEREST(S): This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model.

STUDY QUESTION: Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER: LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY: Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION: This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE: LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

Ovarian damage from chemotherapy and current approaches to its protection.

BACKGROUND: Anti-cancer therapy is often a cause of premature ovarian insufficiency and infertility since the ovarian follicle reserve is extremely sensitive to the effects of chemotherapy and radiotherapy. While oocyte, embryo and ovarian cortex cryopreservation can help some women with cancer-induced infertility achieve pregnancy, the development of effective methods to protect ovarian function during chemotherapy would be a significant advantage. OBJECTIVE AND RATIONALE: This paper critically discusses the different damaging effects of the most common chemotherapeutic compounds on the ovary, in particular, the ovarian follicles and the molecular pathways that lead to that damage. The mechanisms through which fertility-protective agents might prevent chemotherapy drug-induced follicle loss are then reviewed. SEARCH METHODS: Articles published in English were searched on PubMed up to March 2019 using the following terms: ovary, fertility preservation, chemotherapy, follicle death, adjuvant therapy, cyclophosphamide, cisplatin, doxorubicin. Inclusion and exclusion criteria were applied to the analysis of the protective agents. OUTCOMES: Recent studies reveal how chemotherapeutic drugs can affect the different cellular components of the ovary, causing rapid depletion of the ovarian follicular reserve. The three most commonly used drugs, cyclophosphamide, cisplatin and doxorubicin, cause premature ovarian insufficiency by inducing death and/or accelerated activation of primordial follicles and increased atresia of growing follicles. They also cause an increase in damage to blood vessels and the stromal compartment and increment inflammation. In the past 20 years, many compounds have been investigated as potential protective agents to counteract these adverse effects. The interactions of recently described fertility-protective agents with these damage pathways are discussed. WIDER IMPLICATIONS: Understanding the mechanisms underlying the action of chemotherapy compounds on the various components of the ovary is essential for the development of efficient and targeted pharmacological therapies that could protect and prolong female fertility. While there are increasing preclinical investigations of potential fertility preserving adjuvants, there remains a lack of approaches that are being developed and tested clinically.

Analysis of programmed cell death in mouse fetal oocytes.

We report a short-term culture system that allows to define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pan-caspase inhibitors Z-VAD or caspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture. These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.

Experimental approaches to the study of primordial germ cell lineage and proliferation.

New information regarding primordial germ cell (PGC's) segregation and proliferation over the last decade is reviewed. Advances have been obtained in the mouse but current knowledge of human PGC's remains scant. Questions still fully or partially unresolved about the emergence of the germline in mammals are addressed. (i) When and where is the germ line set aside in the embryo? (ii) How is the germ line segregated from the somatic lineages? (iii) Which factors guide PGC's to the gonadal ridges? (iv) Which factors regulate PGC's proliferation? The main purpose of this review is to outline the information obtained using mainly in vitro culture systems about two aspects of these processes namely the segregation of PGC's and their proliferation.

Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus.

Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by approximately 2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This "FAAH activator" was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A(2), C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B(4) or prostaglandins E(2) and F(2)alpha had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated.

A comparative study of cytotoxic effects of N-ethyl-N-nitrosourea, adriamycin, and mono-(2-ethylhexyl)phthalate on mouse primordial germ cells.

Several strategies for the assessment of reproductive toxicity of chemical compounds has have been proposed. In the present work, we devised experimental in vitro assays to test the effect of potential toxicants on proliferating primordial germ cells (PGCs) in vitro using recently developed methods for isolation and culture of mouse PGCs. Primordial germ cells are the embryonic precursors of gametes of the adult that carry the genome from generation to generation. Any damage or mutations caused to these cells by potential toxicants might impair normal reproduction and be transmitted to next generation. Three representative compounds, N-ethyl-N-nitrosourea (ENU), adriamycin (ADR), and mono-(2-ethylhexyl)phthalate (MEHP), toxic to different targets and known to affect germ cell development and impair fertility, were tested on PGCs in culture using three different experimental protocols. Survival and growth of PGCs and their ability to adhere to cell monolayers, were taken as endpoints for drug effects. For each compound, sublethal and acute toxicity doses were determined. In addition, information about the mechanisms of action of these compounds on PGCs was obtained. Whereas the effects of ENU and ADR on PGCs were attributable to growth inhibition and apoptosis induction, MEHP affected PGC adhesion to somatic cells without significantly altering their growth and survival. The results of our in vitro tests were not always exactly predictive of the effects of the tested compounds on PGCs in vivo, determined in parallel experiments in which pregnant mice were exposed to the same compounds. Nevertheless, they can provide information on the sensitivity of PGCs to the direct action of drugs or the mechanisms of action of such agents.