MedicalResearch.com Interview with:
Paola Grimaldi, PhD
Associate Professor of Anatomy
Department of Biomedicine and Prevention,
School of Medicine, University of Rome Tor Vergata
Rome, Italy
MedicalResearch.com: What is the background for this study? What are the main findings?Dr. Grimaldi: Our previous studies reported that mouse mitotic germ cells, spermatogonia, express type 2 cannabinoid receptor (CB2) and its stimulation promoted differentiation and meiotic entry of these cells in vitro. In this study we demonstrate that CB2 plays a role of in regulating the correct progression of spermatogenesis in vivo and we found that the use of exogenous agonist or antagonist of this receptor disrupts the normal differentiation of germ cells. This suggests that a basal and finely regulated level of endocannabinoids in male germ cells activate CB2, thus maintaining the homeostasis of spermatogenesis.
Another important novelty of our study is that CB2 activation in developing germ cells determines the appearance of modifications in DNA-bound proteins, which are known to impact on gene expression and inheritance of specific traits in developing germ cells. An exciting idea could be that these modifications might be maintained in the mature spermatozoa and transmitted to the offspring.
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MedicalResearch.com Interview with:
Dr. Kieron Barclay PhD
Department of Social Policy
London School of Economics
MedicalResearch.com: What is the background for this study? What are the main findings?
Dr. Barclay: Mean age at childbearing has been increasing in most countries in the OECD since the early 1970s. A wealth of research has shown that childbearing at advanced ages is associated with greater difficulty in terms of getting pregnant, higher rates of miscarriage, stillbirth, and increased risk of poor peri-natal outcomes such as pre-term birth and low birth weight. Studies also indicate that the offspring of older mothers have a greater risk of Alzheimer’s disease, diabetes, and mortality in adulthood. However, from the perspective of any individual woman, delaying childbearing to an older age necessarily also means that she will give birth in a later birth year. The last 40 to 50 years have seen substantial improvements in educational opportunities, and better public health conditions and medical knowledge. As a result, these positive secular trends may outweigh or counterbalance the negative effects of reproductive aging for the child.
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MedicalResearch.com Interview with:
Frida Lundberg | PhD Student
Dept. of Medical Epidemiology and Biostatistics
Karolinska InstitutetMedical Research: What is the background for this study?
Response: Fertility treatments involve stimulation with potent hormonal drugs that increase the amount of the sex hormones estrogen and progesterone. These hormones have been linked to breast cancer risk. Further, as these treatments are relatively new, most women who have gone through them are still below the age at which breast cancer is usually diagnosed. Therefore we wanted to investigate if infertility and fertility treatments influences mammographic breast density, a strong marker for breast cancer risk that is also hormone-responsive.
Medical Research: What are the main findings?
Response: We found that women with a history of infertility had higher absolute dense volume than other women. Among the infertile women, those who had gone through controlled ovarian stimulation (COS) had the highest absolute dense volume. The results from our study indicate that infertile women, especially those who undergo COS, might represent a group with an increased risk of breast cancer. However, the observed difference in dense volume was relatively small and has only been linked to a modest increase in breast cancer risk in previous studies. As the infertility type could influence what treatment the couples undergo, the association might also be due to the underlying infertility rather than the treatment per se.
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MedicalResearch.com Interview with:
Marcelo L. Urquia PhD
PhD, MSc, Mg Public Health, BA
Scientist, Li Ka Shing Knowledge Institute
St. Michael’s Hospital
Assistant Professor, Dalla Lana School of Public Health, University of TorontoMedicalResearch.com: What is the background for this study? What are the main findings?
Dr. Urquia: In most populations the sex ratio at birth, that is, ratio of male newborns to female newborns, is about 103 to 107 males per 100 females. This is well established and does not substantially vary according to whether a woman had one or two previous children of the same sex, as each pregnancy is an independent event. However, it is known that several parts of Asia characterize for having son-biased sex ratios at birth. As countries from Asia, such as India and China are the top contributors of births to immigrant women in Canada, we wanted to verify whether son-biased sex ratios were present in Canada. Since induced abortion following prenatal sex determination using ultrasonography has been hypothesized to be a major mechanism that may explain the distorted sex ratios observed in Asia, we also studied the connection between the probability of having boys after induced abortions.
In our first study entitled “Sex ratios after induced abortion” published in CMAJ (http://www.cmaj.ca/lookup/doi/10.1503/cmaj.151074), which used Ontario health care records, we found that sex ratios among Canadian-born women in Ontario were within the expected, irrespective of birth order. The sex ratio among immigrant women from India with two prior girls was 196 males per 100 females for the third live birth. Among Indian immigrant women with two prior daughters the sex ratio increased to 326 males per 100 females if they have had induced abortions preceding the third birth, to 409 males per 100 females if they have had more than one induced abortion since the last newborn child, and to 663 males per 100 females if they have had at least one preceding abortion after 14 weeks of gestation (when the sex of the fetus can be accurately estimated by ultrasonography). These findings suggest that among Indian immigrants to Ontario induced abortions of female fetuses are much more common than induced abortions of male fetuses, which helps explain the deficit in the expected number of female newborns.
In our companion paper entitled “Variations in male-female infant ratios among births toCanadian- and Indian-born mothers, 1990-2011: a population-based register study” and published in CMAJ Open (insert URL), we used national birth certificate data and verified that the patterns observed in Ontario are very likely to apply to all Canadian provinces. Moreover, son-biased sex ratios at birth among Indian immigrants have existed in Canada since the early 1990’s. The deficit in the expected number of girls to Indian immigrants over the last two decades in Canada is in between 3211 to 5921.
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MedicalResearch.com Interview with:Sheree L. Boulet, DrPH, MPH
Division of Reproductive Health
Centers for Disease Control and Prevention
Atlanta, GeorgiaMedicalResearch.com: What is the background for this study?
Dr. Boulet: Findings from some studies have suggested that children conceived with assisted reproductive technology (ART) have increased risks of birth defects compared with spontaneously conceived children. Many of these studies were limited by a small sample size and were unable to assess risks associated with specific ART procedures.
MedicalResearch.com: What are the main findings?
Dr. Boulet: We found that singleton infants conceived using assisted reproductive technology were 1.4 times more likely to have a non-chromosomal birth defect compared with other infants, and the risks were highest for gastrointestinal and musculoskeletal defects. However, when our study was restricted to only ART-conceived infants, no single procedure substantially increased the risk for birth defects. This suggests that the higher risk of birth defects may be due to underlying issues related to infertility, rather than to ART itself.
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MedicalResearch.com Interview with:
Anders Rehfeld MD, PhD Student
University of CopenhagenFaculty of Health and Medical Sciences
Department of Cellular and Molecular Medicine
Copenhagen, Denmark
MedicalResearch.com: What is the background for this study? What are the main findings?
Dr. Rehfeld: Human fertility is declining in many areas of the world and the reason is largely unknown.
Our study shows that 44% tested chemical UV filters can induce calcium signals in human sperm cells, thereby mimicking the effect of progesterone and possibly interfering with the fertilizing ability of human sperm cells. Progesterone-induced calcium signaling, and the sperm functions it triggers, is absolutely essential for the human sperm cell to normally fertilize the human egg.
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MedicalResearch.com Interview with:
[caption id="attachment_22896" align="alignleft" width="175"] Dr. Eric Chow[/caption]
Eric J. Chow, MD, MPH
Hematology-Oncology
Attending physician, Seattle Childrens Hospital
Assistant professor of Pediatrics,...
MedicalResearch.com Interview with:
Melissa Miller, PhD
Postdoctoral fellow at both UC Berkeley and UC San FranciscoMedicalResearch.com: What is the background for this study? What are the main findings?
Dr. Miller: This work builds on years of observed, but unexplained, phenomena within sperm cells which respond almost instantaneously to the presence of the steroid hormone progesterone. Typically, steroid signaling occurs through a long, slow process that involves the modification of gene amount within a cell. However, there is an alternative mechanism that is not well understood that works differently and is termed non-genomic progesterone signaling. We found that progesterone in human sperm cells binds to a protein called ABHD2 and activates its activity to clear the cell of the endogenous cannabinoid 2AG. 2AG is an inhibitor of sperm activation and its removal from the cellular membrane allows the sperm cells to change its motility so that it may reach and fertilize the egg. Men who’s sperm is unable to undergo this progesterone activated motility change are infertile.
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MedicalResearch.com Interview with:
Germaine M. Buck Louis, Ph.D., M.S.
Office of the Director
Division of Intramural Population Health Research
Eunice Kennedy Shriver National Institute of Child Health and Human Development
Rockville, Maryland 20852.
MedicalResearch.com: What is the background for this study? What are the main findings?
Response: To understand the association between couples’ lifestyles and risk of pregnancy loss. Couples were recruited upon discontinuing contraception to try for pregnancy and followed daily for up to one year of trying or until pregnancy. Pregnant women were followed daily for 7 weeks following conception then monthly.
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MedicalResearch.com Interview with:
Kutluk Oktay, MD, PhD.
Professor of Obstetrics & Gynecology, Medicine, and Cell Biology & Anatomy
Director, Division of Reproductive Medicine & Institute for Fertility Preservation
Innovation Institute for Fertility and In Vitro Fertilization
New York Medical College, Valhalla, NY
Medical Research: What is the background for this study? What are the main findings?
Dr.Oktay: Cancer treatments cause infertility and early menopause in a growing number of young women around the world and US. One of the strategies to preserve fertility, which was developed by our team, is to cryopreserve ovarian tissue before chemotherapy and later transplant it back to the patient when they are cured of the cancer and ready to have children. However, success of ovarian transplantation has been limited due to limitation in blood flow to grafts. In this study we described a new approach which seems to improve graft function. The utility of an extracellular tissue matrix and robotic surgery seems to enhance graft function. With this approach both patients conceived with frozen embryos to spare and one has already delivered.(more…)
MedicalResearch.com Interview with
Dr. Norbert Gleicher, MD, FACOG, FACS
Founder, Center for Human Reproduction
Background: What’s My Fertility and the Center for Human Reproduction in New York, have announced the first screening for Premature Ovarian Aging (POA) in young women, based on new research that the FMR1 gene can be predictive of POA.Medical Research: What is Premature Ovarian Aging (POA)?How does POA differ from Premature Ovarian Failure?Dr. Gleicher: Premature Ovarian Aging (POA) is a condition that causes a young woman’s ovaries to age faster than normal. It affects roughly 10% of all women, regardless of race or ethnic background. POA typically causes no symptoms until the ovarian reserve is already very low.
Women are born with all their egg cells (called oocytes). Scientists refer to this as a woman’s original “ovarian reserve.” From birth on, significant numbers of these eggs are constantly lost until menopause. As women age, their ovarian reserve, therefore, depletes and fertility declines.
In most women, fertility begins to decline around age 35 – but for women at risk for POA, fertility declines can begin as early as in their teens or 20s. POA in early stages typically has no symptoms. Most women until now, therefore, are usually only diagnosed after troubles conceiving become apparent, which brings them to a fertility specialist. At that point, most require stressful and costly infertility treatment to have children.
Premature Ovarian Failure (POF), sometimes also called premature menopause of primary ovarian insufficiency (POI) is the end stage of POA, when women reach menopause under age 40. Fortunately, this happens only to 10% of the 10% of women with Premature Ovarian Aging, - which means to 1% of the total female population. In those unfortunate few, even routine IVF can usually no longer help, and most of these women will only conceive with use of young donor eggs.(more…)
MedicalResearch.com Interview with:
Rajiv McCoy, PhD
Dept. of Genome Sciences
Univ. of Washington
Medical Research: What is the background for this study? What are the main findings?Dr. McCoy: Aneuploidy—the inheritance of extra or missing chromosomes compared to the typical 46-chromosome set—is extremely common in human embryos. The vast majority of aneuploidies result in preclinical pregnancy loss, often before the pregnancy is even recognized by the mother. This is thought to be the primary reason why only ~30% of all conceptions result in successful live birth.
Many aneuploidies arise during egg formation, with the frequency increasing with maternal age. In addition to meiotic errors, a large proportion of aneuploidies affecting cleavage-stage embryos are mitotic in origin, arising during the initial post-fertilization cell divisions. These initial divisions are controlled by machinery contributed by the mother in the egg (before the embryo's genome has been activated). While these mitotic errors are frequent in cleavage-stage embryos, we found that they are rare in embryos at day-5 of development (the blastocyst stage), suggesting that embryos and/or cells with extensive mitotic errors do not survive to day 5. We discovered that some women have a greater propensity to produce embryos with mitotic errors than others, and our idea was that maybe differences in the mitotic machinery could help explain this.
Using data from in vitro fertilized embryos screened by our collaborators at Natera, we found that women who have a particular version of a gene called PLK4 tend to produce more aneuploid embryos, regardless of age. This genetic variant is actually very common—more than half of people carry at least one copy—and is present in nearly all populations. PLK4 has a well-known role in ensuring the proper distribution of chromosomes. We also found that patients referred for embryo screening due to previous IVF failure had higher rates of mitotic error, which underscores the clinical importance of this form of whole-chromosome abnormality.
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MedicalResearch.com Interview with:
Audrey J. Gaskins, Sc.D. Postdoctoral Fellow
Department of Nutrition
Harvard T.H. Chan School of Public Health
Boston, MA 02115
Medical Research: What is the background for this study? What are the main findings?
Dr. Gaskins: Infertility, defined as the inability to conceive after 12 months of unprotected intercourse, is a common reproductive disorder affecting ~15% of couples who attempt to become pregnant. Assisted reproductive technologies (ART), which include in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI), have become the main treatment modalities for couples facing infertility. Pre-conceptional folate and vitamin B12 have been linked to many beneficial early pregnancy outcomes among couples undergoing assisted reproductive technologies treatment in Europe but mixed results have been found in regards to clinical pregnancy and live birth rates. Therefore, we sought to investigate whether higher levels of serum folate and vitamin B12 could increase reproductive success in a cohort of women undergoing assisted reproductive technologies at an academic medial center in the United States.
We found that high concentrations of folate and vitamin B12 in serum are associated with increased chance of live birth following assisted reproduction. Moreover, women with higher concentrations of both serum folate and vitamin B12 had the greatest likelihood of reproductive success. Analysis of intermediate endpoints suggests that folate and vitamin B12 may exert their favorable effects on pregnancy maintenance following implantation.
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MedicalResearch.com Interview with: Camilla Sandal Sejbaek PhD
Department of Public Health
University of Copenhagen
Medical Research: What is the background for this study? What are the main findings?Response: Previous literature have shown ambiguous results when investigating the association between becoming a mother and depression among women in fertility treatment. Small questionnaire-based studies with self-reported depression have shown that women in unsuccessful fertility treatment had a higher risk of depressive symptoms compared to women in successful fertility treatment. Two larger register-based studies using clinical depression (depression diagnosed at the psychiatric hospitals) have shown that women becoming a mother are at increased risk of clinical depression.
Our findings, from a large register-based study with about 41,000 women in assisted reproductive technology (ART) treatment, showed that women WHO became mothers had a higher risk of clinical depression compared to women in ART treatment WHO did not become mothers. The risk of clinical depression were more than five-fold higher within the first 6 weeks after becoming a mother to a live-born child.
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MedicalResearch.com Interview with:
Vitaly A Kushnir MD
The Center for Human Reproduction
New York, NY 10021
Medical Research: What is the background for this study? What are the main findings?
Dr. Kushnir: In January 2013, the American Society for Reproductive Medicine declared the technique of oocyte cryopreservation no longer experimental, although they body did call for further study.
Vitaly A. Kushnir, M.D., of the Center for Human Reproduction, and colleagues used 2013 data from 380 U.S fertility centers to compare live birth and cycle cancellation rates using either fresh or cryopreserved donor oocytes.
The study found roughly 20 percent of donor cycles used cryopreserved oocytes and 80 percent fresh oocytes. Of those embryos transferred, 56 percent that started as fresh oocytes resulted in live births compared to just 47 percent of those that started as cryopreserved oocytes. (more…)
MedicalResearch.com Interview with:
Prof. Dr. med. Christian F. Poets
Neonatologie, Univ.-Klinikum Tübingen
Tübingen Germany
Medical Research: What is the background for this study?
Prof. Poets: Episodes of intermittent hypoxemia (lack of oxygen) and bradycardia (slow heart rate) are common in very preterm infants and often a subject of considerable concern. However, up to now there has been a lack of knowledge as to how often or how long such episodes may occur without increasing an infant’s risk for impaired development or even death.
In this study, we utilized long-term recordings (lasting 8-12 weeks) of oxygen saturation and heart rate obtained as part of the Canadian Oxygen Trial (COT), a large study performed in extremely immature infants and comparing a higher with a lower oxygen saturation target range (85-89 vs. 91-95% oxygen saturation measured by pulse oximetry). For this secondary analysis, we wanted to test the hypotheses that spending a high proportion of time at an oxygen saturation below 80% or a pulse rate <80 beats per minute increases the risk of the following adverse outcomes:
Death after reaching a post-menstrual age of 36 weeks (i.e. 4 weeks before their due date) or disability, determined at 18-22 months corrected age and defined as motor impairment, cognitive or language delay, severe hearing loss, or bilateral blindness;
Motor impairment (determined at 18-22 months corrected age);
Cognitive or language delay (determined at 18-22 months corrected age);
Severe retinopathy of prematurity.
Medical Research: What are the main findings?Prof. Poets: Analyzable recordings and outcome data were available for 1019 infants, of which the least affected 10% spent 0.4%, and the most affected infants 13.5% of the time at an oxygen saturation <80%. We found that the risk to develop all of the adverse outcomes mentioned above increased with the percentage of time spent at an oxygen saturation below 80%, but this was true only for hypoxemic events lasting for at least 1 minute. Episodes with a low heart rate (in the absence of concomitant hypoxemia) were not associated with an increased risk of an adverse outcome. Interestingly, hypoxemic events occurring in infants originally randomized to the higher oxygen group in the original COT study were associated with a stronger increase in the risk of death or disability than such episodes occurring infants randomized to the lower oxygen saturation target range. (more…)
MedicalResearch.com Interview with:
Dr. Audrey J Gaskins
Department of Nutrition
Harvard T.H. Chan School of Public Health
Boston, MA
Medical Research: What is the background for this study? What are the main findings?
Dr. Gaskins: Previous studies have linked shift work, long working hours, and physical factors to an increased risk of menstrual cycle disturbances, spontaneous abortion, preterm birth, and low birth weight; however the association with fecundity is inconsistent. Several papers have also reviewed the occupational exposures of health care workers and concluded that reproductive health issues are a concern. Therefore we sought to determine the extent to which work schedules and physical factors were associated with fecundity in a large cohort of nurses. Women who work in an industry that requires them to work from a height or even lift heavy objects requires them to undertake training which guides them though the effective stages on how to work safely at heights. Without the right training, this sort of work can become very dangerous.
Our main findings were that that working >40 hours per week and moving or lifting a heavy load >15 times per day (including repositioning or transferring patients) were associated with reduced fecundity in our cohort of female nurses planning pregnancy. However, all other factors such as frequency of night work, duration of rotating and non-rotating night shifts, and time spent walking or standing at work were not significantly associated with fecundity in this cohort.
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MedicalResearch.com Interview with:
Carmen J. Williams, M.D., Ph.D. Principal Investigator
National Institute of Environmental Health
Medical Research: What is the background for this study? Dr. Williams: G-protein coupled receptors are used by almost all cells to receive signals from the outside of the cell and transduce these signals into actions within the cell. There are hundreds of these receptors, but many of them link to a protein named Gq to transmit their signals to other cellular proteins. Gq is found in most cells, including eggs, and activating Gq protein is one way to artificially activate the egg to begin developing into an embryo, even though it is not the way sperm normally do this. In fact, if the egg is activated before the sperm arrives it prevents the sperm from binding and fusing with the egg, so fertilization cannot take place. As a result, we thought that a mechanism might be in place within eggs to prevent them from being activated prematurely by signals that could activate Gq by triggering G-protein coupled receptors. RGS2 was a good candidate for this function because it binds to Gq in a way that prevents Gq from transmitting signals to other cellular proteins.
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MedicalResearch.com Interview with:
Stephen A. Krawetz, Ph.D.
Associate Director C.S. Mott Center for Human Growth and Development,
Charlotte B. Failing Professor of Fetal Therapy and Diagnosis,
Department of Obstetrics and Gynecology,
Center for Molecular Medicine and Genetics,
Wayne State University School of Medicine,
Detroit, MI, 48201
Medical Research: What is the background for this study? What are the main findings?
Dr. Krawetz: The current study developed over approximately the past 20 years of work in my laboratory. In the mid 1990s, along with David Miller, we independently discovered that sperm contain RNA. This was followed by our joint publication in The Lancet that began to describe the RNAs in normal fertile males along with our paper in Nature that showed that RNA was delivered to the oocyte at fertilization. Following these studies we assessed the ability of RNAs to be used as markers of morphologically abnormal sperm (teratozoospermia). My laboratory then had the opportunity to explore the complexity of the population of sperm RNAs using Next Generation Sequencing. We recently began the translation of this work from the bench to bedside which takes us to the current paper in Science Translational Medicine that was a multi-institutional collaborative effort. Members of the team include Dr. Meritxell Jodar, Edward Sendler, Robert Goodrich, from my laboratory, along with Dr. Clifford L. Librach, Dr. Sergey I. Moskovtsev, and Sonja Swanson - CReATe Fertility Center, University of Toronto; Dr. Russ Hauser -Harvard University and Dr. Michael P. Diamond, Georgia Regents University. Here we tackled the issue of idiopathic infertility, that is, unknown infertility, since the couple appears normal in all respects. We specifically framed our study as the contribution of the male and female as a couple towards the birth of a healthy child focusing on male idiopathic infertility within the setting of a Reproductive Clinic. Representative publications from my laboratory that outline this part of my research program appear below.
1) Jodar, M., Sendler, E., Moskovtsev, S. Librach, C., Goodrich, R., Swanson, S., Hauser, R., Diamond, M. and Krawetz, S.A. (2015) Absence of sperm RNA elements correlates with idiopathic male infertility. Science Translational Medicine, 7(295):295re6.
2) Sendler, E., Johnson, G.D., Mao, S., Goodrich, R.J., Diamond, M.P., Hauser, R., and Krawetz, S.A. (2013) Stability, Delivery and Functions of Human Sperm RNAs at Fertilization. Nucleic Acids Research 41:4104-4117. PMID: 23471003
3) Platts, A.E., Dix, D. J., Chemes, H.E., Thompson, K.E., Goodrich, R., Rockett, J. C., Rawe, V.Y., Quintana, S., Diamond, M.P., Strader, L.F. and Krawetz, S.A. (2007) Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs. Human Molecular Genetics. 16:763-773. PMID: 17327269
4) Ostermeier, G.C., Miller, D., Huntriss, J.D., Diamond, M.P. and Krawetz, S.A. (2004) Delivering spermatozoan RNA to the oocyte. Nature 429:154. PMID: 15141202
5) Ostermeier, G.C., Dix, D.J., Miller, D., Khatri, P. and Krawetz, S.A. (2002) Spermatozoal RNA profiles of normal fertile men. The Lancet. 360:773-777. PMID: 12241836
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MedicalResearch.com Interview with:
Alexander N Yatsenko, MD, PhD
Assistant Professor,
Department of OBGYN and Reproductive Science,
Magee-Womens Research Institute,
University of Pittsburgh, PA
Pittsburgh, PA 15213
Medical Research: What is the background for this study? What are the main findings?
Dr. Yatsenko: The known causes of male infertility not due to physical obstruction are usually because of sex-chromosome defects, such as deletions of the Y chromosome or duplication of the entire X chromosome in Klinefelter syndrome. Eight times out of 10, conventional genetic testing doesn’t reveal a chromosomal problem and infertility is considered idiopathic. We wanted to try to find other genetic reasons for the problem.
We found a deletion in part of the DNA coding of the testis-expressed gene 11 (TEX11) on the X-chromosome, which men inherit from their mothers. The alteration caused meiotic arrest, meaning the precursor cells could not properly undergo meiosis. We also found similar TEX11 gene mutations and meiotic arrest in two out of 49 men diagnosed with idiopathic azoospermia in Pittsburgh or at a Poland infertility clinic, and in five out of 240 infertile men assessed at a collaborating Andrology clinic in Muenster, Germany. These genetic findings were confirmed on protein level using patients’ testis biopsies. (more…)
MedicalResearch. com Interview with: Michael Eisenberg, MD, PhD
Director, Male Reproductive Medicine and Surgery
Assistant Professor, Department of Urology
Assistant Professor, Department of Obstetrics and Gynecology
Stanford School of Medicine
MedicalResearch: What are the main findings of the study?Dr. Eisenberg: There is an inverse relationship between semen quality and mortality so that as semen quality declines the likliehood of death increases.
(more…)
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