Sacha Krieg

Correction of a pathogenic gene mutation in human embryos

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

VEGF blockade inhibits angiogenesis and reepithelialization of endometrium

Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangio‐genesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2‐neuropilin 1 (VEGFR2‐NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endo‐metrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angio‐genesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.—Fan, X., Krieg, S., Kuo, C. J., Wiegand, S. J., Rabinovitch, M., Druzin, M. L., Brenner, R. M., Giudice, L. C., Nayak, N. R. VEGF blockade inhibits angiogenesis and reepithelialization of endometrium. FASEB J. 22, 3571–3580 (2008)

Clinical guidelines for sperm cryopreservation in cancer patients

No clear clinical guidelines exist on how to counsel male cancer patients about fertility preservation. Detailed counseling is recommended before treatment when issues of collection and storage need to be highlighted. Concern about the quality of sperm collected before and/or after treatment in terms of assisted reproduction is needed, and the potential outcomes should be discussed early as part of cancer survivorship. The discussion should be sensitive and tailored to the ethical situation based on the age of the patient, the severity of the illness, the need to initiate treatment, and genetic risk. Cryopreservation should be attempted/achieved before cancer treatment is initiated. Cryopreservation should not be performed during treatment or for some time after treatment because of the chromosomal and structural damage to sperm from cancer treatment. Contraception should be instigated during this period.

A Unique Downstream Estrogen Responsive Unit Mediates Estrogen Induction of Proteinase Inhibitor-9, a Cellular Inhibitor of IL-1β- Converting Enzyme (Caspase 1)

Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss

Recurrent pregnancy loss (RPL) occurs in ∼5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.

Immune Function and Recurrent Pregnancy Loss

Recurrent pregnancy loss (RPL), defined as two or more consecutive miscarriages, is attributable to multiple causes. However, in 50% of cases no known cause is found. Although endometritis is a known cause of miscarriage, other inflammatory processes may play a role in idiopathic, recurrent loss. The fetoplacental unit evades rejection by the maternal immune system by poorly understood mechanisms. Despite this seemingly immune-privileged state for the fetus, human implantation requires inflammatory mediators for attachment and implantation. This review describes how the immune system must simultaneously permit and restrict trophoblastic invasion for healthy implantation and maintenance of pregnancy. Included in this review is a detailed description of the immune milieu in the decidua and abnormalities that are associated with RPL. Finally, autoimmune states associated with RPL and their treatment in an obstetrical setting are reviewed.

Dynamic Regulation of Wnt7a Expression in the Primate Endometrium: Implications for Postmenstrual Regeneration and Secretory Transformation

Despite the vital physiological role of endometrial regeneration during the menstrual cycle and the various pathological implications of abnormal growth of endometrial epithelial cells, the local factors and regulatory mechanisms involved in endometrial regeneration and growth have not been well characterized. Here, we examine the pattern, hormone dependence, and potential functions of Wnt7a (wingless-type MMTV integration site family member 7a), which is known to play a critical role in the formation of the mouse endometrial epithelium during embryonic development, in both human and artificially cycling rhesus macaque endometrium, and using a potent Wnt-antagonist in a mouse model of endometrial regeneration. Wnt7a transcript levels were examined using quantitative real-time PCR and in situ hybridization, and immunohistochemistry was performed to detect Ki-67 and 3,5-bromodeoxyuridine. Stringent, fully conditional Wnt inhibition was achieved by adenoviral expression of Dickkopf-1 during artificial endometrial regeneration in mice. In macaques, Wnt7a expression was confined to the newly formed luminal epithelium (LE) and upper glands during the postmenstrual repair phase. The signal increased in the LE during the proliferative phase but decreased in the upper glands and was undetectable in the glands by the late proliferative phase. Interestingly, Wnt7a was completely suppressed in the LE and remained undetectable in other cell types after 7 d of progesterone treatment. The pattern of Wnt7a expression in the human endometrium was similar to that in macaques. Blockade of Wnt signaling during endometrial regeneration in mice resulted in a dramatic delay in reepithelialization and degeneration of glands and LE. These results strongly suggest, for the first time, a role for Wnt7a in postmenstrual regeneration and proliferation of endometrial glands and LE in primates, and its dramatic suppression by progesterone is likely essential for secretory transformation of the epithelium.

Variable expression of soluble fms-like tyrosine kinase 1 in patients at high risk for preeclampsia.

Objective. To explore angiogenic factor differences in preeclamptic patients according to the absence or presence of underlying vascular disease. Methods. We prospectively compared serum soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin, and placental growth factor (PlGF) from 41 normal-risk and 32 high-risk (preexisting conditions) subjects at serial gestational ages. Results. Median sFlt1 was lower at delivery in preeclamptic patients with underlying chronic hypertension and/or chronic proteinuria (5115 pg/ml) compared with normal risk preeclamptic patients (16375 pg/ml). PlGF was consistently low in patients who developed preeclampsia. Conclusions. Effects of sFlt1 may be contextual, varying according to the health or disease state of vascular endothelium.

Environmental exposure to endocrine-disrupting chemicals and miscarriage

Establishment of early pregnancy is the result of complex biochemical interactions between the decidua and blastocyst. Any alteration in this chemical dialogue has the potential to result in adverse pregnancy outcomes including miscarriage. Sporadic miscarriage is the most common complication of pregnancy and can be caused by multiple factors. While the most common cause of miscarriage is genetic abnormalities in the fetus, other contributing factors certainly can play a role in early loss. One such factor is environmental exposure, in particular to endocrine-disrupting chemicals, which has the potential to interfere with endogenous hormone action. These effects can be deleterious, especially in early pregnancy when the hormonal milieu surrounding implantation is in delicate balance. The purpose of this paper is to review the current evidence on the role of environmental toxins in reproduction.

NORMAL PREGNANCY AFTER TETRAPLOID KARYOTYPE ON TROPHECTODERM BIOPSY

OBJECTIVE To report a case of successful pregnancy after trophectoderm biopsy and fluorescence in situ hybridization (FISH) revealed a tetraploid karyotype. DESIGN Case report. SETTING A university medical center. PATIENT(S) An infertility patient desiring trophectoderm biopsy on frozen blastocysts to facilitate preimplantation genetic screening. INTERVENTION(S) Frozen blastocysts were thawed on the evening before transfer. Trophectoderm biopsy was performed the following morning. FISH results were available the same day, and two embryos with tetraploid results were transferred. MAIN OUTCOME MEASURE(S) Chorionic villus sample (CVS) and newborn exam. RESULT(S) Normal diploid CVS result and a healthy male infant. CONCLUSION(S) Although multiple cells can be analyzed using trophectoderm biopsy, abnormalities in the trophectoderm may not be present in the inner cell mass.