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dr Firman Abdullah SpOG / OBGYN

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Thursday, May 14, 2009

Health and Nutritional Benefits from Coconut Oil: An Important Functional Food for the 21st Century


A New Look at Coconut Oil
By Mary G. Enig, Ph.D.

Health and Nutritional Benefits from Coconut Oil: An Important Functional Food for the 21st Century
Presented at the AVOC Lauric Oils Symposium, Ho Chi Min City, Vietnam, 25 April 1996

Coconut oil has a unique role in the diet as an important physiologically functional food. The health and nutritional benefits that can be derived from consuming coconut oil have been recognized in many parts of the world for centuries. Although the advantage of regular consumption of coconut oil has been underappreciated by the consumer and producer alike for the recent two or three decades, its unique benefits should be compelling for the health minded consumer of today. A review of the diet/heart disease literature relevant to coconut oil clearly indicates that coconut oil is at worst neutral with respect to atherogenicity of fats and oils and, in fact, is likely to be a beneficial oil for prevention and treatment of some heart disease. Additionally, coconut oil provides a source of antimicrobial lipid for individuals with compromised immune systems and is a nonpromoting fat with respect to chemical carcinogenesis.

I. Introduction
Mr. Chairman and members of the ASEAN Vegetable Oils Club, I would like to thank you for inviting me to participate in this Lauric Oils Symposium. I am pleased to have the opportunity to review with you some information that I hope will help redress some of the anti-tropical oils rhetoric that has been so troublesome to your industry.

I will be covering two important areas in my presentation. In the first part, I would like to review the history of the major health challenge facing coconut oil today. This challenge is based on a supposed negative role played by saturated fat in heart disease. I hope to dispel any acceptance of this notion with the information I will present to you today. I will show you how both animal studies and human studies have exonerated coconut oil of causing the problem.

In the second part of my talk I will suggest some new directions where important positive health benefits are seen for coconut oil. These benefits stem from coconut oil's use as a food with major antimicrobial and anticancer benefits. I will present to you some of the rationale for this effect and some of the supporting literature.

The health and nutritional benefits derived from coconut oil are unique and compelling. Although the baker and food processor have recognized the functional advantages of coconut oil in their industries, over most competing oils, for many years, I believe these benefits are underappreciated today by both the producer and the consumer. It is time to educate and reeducate all t hose who harbor this misinformation.

Historically, coconuts and their extracted oil have served man as important foods for thousands of years. The use of coconut oil as a shortening was advertised in the United States in popular cookbooks at the end of the 19th century. Both the health-promoting attributes of coconut oil and those functional properties useful to the homemaker were recognized 100 years ago. These same attributes, in addition to some newly discovered ones, should be of great interest to both the producing countries as well as the consuming countries.

II. Origins of the Diet/Heart Hypothesis
Although popular literature of epidemiological studies usually attribute an increased risk of coronary heart disease (CHD) to elevated levels of serum cholesterol, which in turn are thought to derive from a dietary intake of saturated fats and cholesterol. But, saturated fats may be considered a major culprit for CHD only if the links between serum cholesterol and CHD, and between saturated fat and serum cholesterol are each firmly established. Decades of large-scale tests and conclusions therefrom have purported to establish the first link. In fact, this relationship has reached the level of dogma. Through the years metabolic ward and animal studies have claimed that dietary saturated fats increase serum cholesterol levels, thereby supposedly establishing the second link. But the scientific basis for these relationships has now been challenged as resulting from large-scale misinterpretation and misrepresentation of the data. (Enig 1991, Mann 1991, Smith 1991, Ravnskov 1995)

Ancel Keys is largely responsible for starting the anti-saturated fat agenda in the United States. From 1953 to 1957 Keys made a series of statements regarding the atherogenicity of fats. These pronouncements were:

"All fats raise serum cholesterol; Nearly half of total fat comes from vegetable fats and oils; No difference between animal and vegetable fats in effect on CHD (1953); Type of fat makes no difference; Need to reduce margarine and shortening (1956); All fats are comparable; Saturated fats raise and polyunsaturated fats lower serum cholesterol; Hydrogenated vegetable fats are the problem; Animal fats are the problem (1957-1959)."
As can be seen, his findings were inconsistent.

What was the role of the edible oil industry in promoting the diet/heart hypothesis?
It is important to realize that at that time (1960s) the edible oil industry in the United States seized the opportunity to promote its polyunsaturates. The industry did this by developing a health issue focusing on Key's anti-saturated fat bias. With the help of the edible oil industry lobbying in the United States, federal government dietary goals and guidelines were adopted incorporating this mistaken idea that consumption of saturated fat was causing heart disease. This anti-saturated fat issue became the agenda of government and private agencies in the US and to an extent in other parts of the world. This is the agenda that has had such a devastating effect on the coconut industry for the past decade. Throughout the 1960s, the 1970s, the 1980s, and the 1990s, the anti-saturated fat rhetoric increased in intensity.

What are some of the contradictions to the hypothesis blaming saturated fat?
Recently, an editorial by Harvard's Walter Willett, M.D. in the American Journal of Public Health (1990) acknowledged that even though

"the focus of dietary recommendations is usually a reduction of saturated fat intake, no relation between saturated fat intake and risk of CHD was observed in the most informative prospective study to date."
Another editorial, this time by Framingham's William P. Castelli in the Archives of Internal Medicine (1992), declared for the record that

"...in Framingham, Mass, the more saturated fat one ate, the more cholesterol one ate, the more calories one ate, the lower the person's serum cholesterol... the opposite of what the equations provided by Hegsted at al (1965) and Keys et al (1957) would predict..."
Castelli further admitted that

"...In Framingham, for example, we found that the people who ate the most cholesterol, ate the most saturated fat, ate the most calories, weighed the least, and were the most physically active."
III. Coconut Oil and the Diet/Heart Hypothesis
For the past several decades you have heard about animal and human studies feeding coconut oil that purportedly showed increased indices for cardiovascular risk. Blackburn et al (1988) have reviewed the published literature of coconut oil's effect on serum cholesterol and atherogenesis and have concluded that when ...[coconut oil is] fed physiologically with other fats or adequately supplemented with linoleic acid, coconut oil is a neutral fat in terms of atherogenicity. After reviewing this same literature, Kurup and Rajmohan (1995) conducted a study on 64 volunteers and found ...no statistically significant alteration in the serum total cholesterol, HDL cholesterol, LDL cholesterol, HDL cholesterol/total cholesterol ratio and LDL cholesterol/HDL cholesterol ratio of triglycerides from the baseline values... A beneficial effect of adding the coconut kernel to the diet was noted by these researchers.

How did coconut oil get such a negative reputation?
The question then is, how did coconut oil get such a negative reputation? The answer quite simply is, initially, the significance of those changes that occurred during animal feeding studies were misunderstood. The wrong interpretation was then repeated until ultimately the misinformation and disinformation took on a life of its own.

The problems for coconut oil started four decades ago when researchers fed animals hydrogenated coconut oil that was purposefully altered to make it completely devoid of any essential fatty acids. The hydrogenated coconut oil was selected instead of hydrogenated cottonseed, corn or soybean oil because it was a soft enough fat for blending into diets due to the presence of the lower melting medium chain saturated fatty acids. The same functionality could not be obtained from the cottonseed, corn or soybean oils if they were made totally saturated, since all their fatty acids were long chain and high melting and could not be easily blended nor were they as readily digestible.

The animals fed the hydrogenated coconut oil (as the only fat source) naturally became essential fatty acid deficient; their serum cholesterol levels increased. Diets that cause an essential fatty acid deficiency always produce an increase in serum cholesterol levels as well as an increase in the atherosclerotic indices. The same effect has also been seen when other essential fatty acid deficient, highly hydrogenated oils such as cottonseed, soybean, or corn oils have been fed; so it is clearly a function of the hydrogenated product, either because the oil is essential fatty acid (EFA) deficient or because of trans fatty acids (TFA).

What about the studies where animals were fed with unprocessed coconut oil?
Hostmark et al (1980) compared the effects of diets containing 10% coconut fat and 10% sunflower oil on lipoprotein distribution in male Wistar rats. Coconut oil feeding produced significantly lower levels (p=<0.05) of pre-beta lipoproteins (VLDL) and significantly higher (p=<0.01) alpha-lipoproteins (HDL) relative to sunflower oil feeding.

Awad (1981) compared the effects of diets containing 14% coconut oil, 14% safflower oil or a 5% "control" (mostly soybean) oil on accumulation of cholesterol in tissues in male Wistar rats. The synthetic diets had 2% added corn oil with a total fat of 16% Total tissue cholesterol accumulation for animals on the safflower diet was six times greater than for animals fed the coconut oil, and twice that of the animals fed the control oil.

A conclusion that can be drawn from some of this animal research is that feeding hydrogenated coconut oil devoid of essential fatty acids (EFA) in a diet otherwise devoid of EFA leads to EFA deficiency and potentiates the formation of atherosclerosis markers. It is of note that animals fed regular coconut oil have less cholesterol deposited in their livers and other parts of their bodies.

What about the studies where coconut oil is part of the normal diet of human beings?
Kaunitz and Dayrit (1992) have reviewed some of the epidemiological and experimental data regarding coconut-eating groups and noted that the available population studies show that dietary coconut oil does not lead to high serum cholesterol nor to high coronary heart disease mortality or morbidity. They noted that in 1989 Mendis et al reported undesirable lipid changes when young adult Sri Lankan males were changed from their normal diets by the substitution of corn oil for their customary coconut oil. Although the total serum cholesterol decreased 18.7% from 179.6 to 146.0 mg/dl and the LDL cholesterol decreased 23.8% from 131.6 to 100.3 mg/dl, the HDL cholesterol decreased 41.4% from 43.4 to 25.4 mg/dl (putting the HDL values below the acceptable lower limit) and the LDL/HDL ratio increased 30% from 3.0 to 3.9. These latter two changes would be considered quite undesirable. As noted above, Kurup and Rajmohan (1995) studied the addition of coconut oil alone to previously mixed fat diets and report no significant difference.

Previously, Prior et al (1981) had shown that islanders with high intake of coconut oil showed no evidence of the high saturated fat intake having a harmful effect in these populations. When these groups migrated to New Zealand however, and lowered their intake of coconut oil, their total cholesterol and LDL cholesterol increased, and their HDL cholesterol decreased.

What about the studies where coconut oil was deliberately fed to human beings?
Some of the studies reported thirty and more years ago should have cleared coconut oil of any implication in the development of coronary heart disease (CHD).

For example, when Frantz and Carey (1961) fed an additional 810 kcal/day fat supplement for a whole month to males with high normal serum cholesterol levels, there was no significant difference from the original levels even though the fat supplement was hydrogenated coconut oil.

Halden and Lieb (1961) also showed similar results in a group of hyperchole-sterolemics when coconut oil was included in their diets. Original serum cholesterol levels were reported as 170 to 370 mg/dl. Straight coconut oil produced a range from 170 to 270 mg/dl. Coconut oil combined with 5% sunflower oil and 5% olive oil produced a range of 140 to 240 mg/dl.

Earlier, Hashim and colleagues (1959) had shown quite clearly that feeding a fat supplement to hypercholesterolemics, where half of the supplement (21% of energy) was coconut oil (and the other half was safflower oil), resulted in significant reductions in total serum cholesterol. The reductions averaged -29% and ranged from -6.8 to -41.2%.

And even earlier, Ahrens and colleagues (1957) had shown that adding coconut oil to the diet of hypercholesterolemics lowers serum cholesterol from, e.g., 450 mg/dl to 367 mg/dl. This is hardly a cholesterol-raising effect.

Bierenbaum et al (1967) followed 100 young men with documented myocardial infarction for 5 years on diets with fat restricted to 28% of energy. There was no significant difference between the two different fat mixtures (50/50 corn and safflower oils or 50/50 coconut and peanut oils), which were fed as half of the total fat allowance; both diets reduced serum cholesterol. This study clearly showed that 7% of energy as coconut oil was as beneficial to the 50 men who consumed it as for the 50 men who consumed 7% of energy as other oils such as corn oil or safflower. Both groups fared better than the untreated controls.

More recently, Sundram et al (1994) fed whole foods diets to healthy normo-cholesterolemic males, where approximately 30% of energy was fat. Lauric acid (C12:0) and myristic acid (C14:0) from coconut oil supplied approximately 5% of energy. Relative to the baseline measurements of the subjects prior to the experimental diet, this lauric and myristic acid-rich diet showed an increase in total serum cholesterol from 166.7 to 170.0 mg/dl (+1.9%), a decrease in low density lipoprotein cholesterol (LDL-C) from 105.2 to 104.4 mg/dl (-0.1%), an increase in high density lipoprotein cholesterol (HDL-C) from 42.9 to 45.6 mg/dl (+6.3%). There was a 2.4% decrease in the LDL-C/HDL-C ratio from 2.45 to 2.39. These findings indicate a favorable alteration in serum lipoprotein balance was achieved when coconut oil was included in a whole food diet at 5% of energy.

Tholstrup et al (1994) report similar results with whole foods diets high in lauric and myristic acids from palm kernel oil. The HDL cholesterol levels increased significantly from baseline values (37.5 to 46.0 mg/dl, P<0.01) and the LDL-C/HDL-C ratios decreased from 3.08 to 2.69. The increase in total cholesterol was from 154.7 (baseline) to 170.9 mg/dl on the experimental diet.

Ng et al (1991) fed 75% of the fat ration as coconut oil (24% of energy) to 83 adult normocholesterolemics (61 males and 22 females). Relative to baseline values, the highest values on the experimental diet for total cholesterol was increased 17% (169.6 to 198.4 mg/dl), HDL cholesterol was increased 21.4% (44.3 to 53.8 mg/dl), and the LDL-C/HDL-C ratio was decreased 3.6% (2.51 to 2.42).

When unprocessed coconut oil is added to an otherwise normal diet, there is frequently no change in the serum cholesterol although some studies have shown a decrease in total cholesterol. For example, when Ginsberg et al provided an "Average American" diet with 2-3 times more myristic acid (C14:0), 4.5 times more lauric acid (C12:0), and 1.2 times more palmitic and stearic acid (C16:0 and C18:0) than their "Mono[unsaturated]" diet and the National Cholesterol Education Program "Step 1" diet, there was no increase in serum cholesterol, and in fact, serum cholesterol levels for this diet group fell approximately 3% from 177.1 mg% to 171.8 mg% during the 22 week feeding trial.

It appears from many of the research reports that the effect coconut oil has on serum cholesterol is the opposite in individuals with low serum cholesterol values and those with high serum values. We see that there may be a raising of serum total cholesterol, LDL cholesterol and especially HDL cholesterol in individuals with low serum cholesterol. On the other hand there is lowering of total cholesterol and LDL cholesterol in hypercholesterolemics as noted above.

Studies that supposedly showed a hypercholesterolemic effect of coconut oil feeding, in fact, usually only showed that coconut oil was not as effective at lowering the serum cholesterol as was the more unsaturated fat being compared. This appears to be in part because coconut oil does not drive cholesterol into the tissues as does the more polyunsaturated fats. The chemical analysis of the atheroma shows that the fatty acids from the cholesterol esters are 74% unsaturated (41% is polyunsaturated) and only 24% are saturated. None of the saturated fatty acids were reported to be lauric acid or myristic acid (Felton et al 1994).

Should coconut oil be used to prevent coronary heart disease?
There is another aspect to the coronary heart disease picture. This is related to the initiation of the atheromas that are reported to be blocking arteries. Recent research is suggestive that there is a causative role for the herpes virus and cytomegalovirus in the initial formation of atherosclerotic plaques and the recloging of arteries after angioplasty. (New York Times 1991) What is so interesting is that the herpes virus and cytomegalovirus are both inhibited by the antimicrobial lipid monolaurin; but monolaurin is not formed in the body unless there is a source of lauric acid in the diet. Thus, ironically enough, one could consider the recommendations to avoid coconut and other lauric oils as contributing to the increased incidence of coronary heart disease.

Perhaps more important than any effect of coconut oil on serum cholesterol is the additional effect of coconut oil on the disease fighting capability of the animal or person consuming the coconut oil.

IV. Coconut Oil and Cancer
Lim-Sylianco (1987) has reviewed 50 years of literature showing anticarcinogenic effects from dietary coconut oil. These animal studies show quite clearly the nonpromotional effect of feeding coconut oil.

In a study by Reddy et al (1984) straight coconut oil was more inhibitory than MCT oil to induction of colon tumors by azoxymethane. Chemically induced adenocarcinomas differed 10-fold between corn oil (32%) and coconut oil (3%) in the colon. Both olive oil and coconut oil developed the low levels (3%) of the adenocarcinomas in the colon, but in the small intestine animals fed coconut oil did not develop any tumors while 7% of animals fed olive oil did.

Studies by Cohen et al (1986) showed that the nonpromotional effects of coconut oil were also seen in chemically induced breast cancer. In this model, the slight elevation of serum cholesterol in the animals fed coconut oil was protective as the animals fed the more polyunsaturated oil had reduced serum cholesterol and more tumors. The authors noted that "...an overall inverse trend was observed between total serum lipids and tumor incidence for the 4 [high fat] groups."

This is an area that needs to be pursued.

V. Coconut Oil Antimicrobial Benefits
I would now like to review for you some of the rationale for the use of coconut oil as a food that will serve as the raw material to provide potentially useful levels of antimicrobial activity in the individual.

The lauric acid in coconut oil is used by the body to make the same disease-fighting fatty acid derivative monolaurin that babies make from the lauric acid they get from their mothers= milk. The monoglyceride monolaurin is the substance that keeps infants from getting viral or bacterial or protozoal infections. Until just recently, this important benefit has been largely overlooked by the medical and nutrition community.

Recognition of the antimicrobial activity of the monoglyceride of lauric acid (monolaurin) has been reported since 1966. The seminal work can be credited to Jon Kabara. This early research was directed at the virucidal effects because of possible problems related to food preservation. Some of the early work by Hierholzer and Kabara (1982) that showed virucidal effects of monolaurin on enveloped RNA and DNA viruses was done in conjunction with the Center for Disease Control of the US Public Health Service with selected prototypes or recognized representative strains of enveloped human viruses. The envelope of these viruses is a lipid membrane.

Kabara (1978) and others have reported that certain fatty acids (e.g., medium-chain saturates) and their derivatives (e.g., monoglycerides) can have adverse effects on various microorganisms: those microorganisms that are inactivated include bacteria, yeast, fungi, and enveloped viruses.

The medium-chain saturated fatty acids and their derivatives act by disrupting the lipid membranes of the organisms (Isaacs and Thormar 1991) (Isaacs et al 1992). In particular, enveloped viruses are inactivated in both human and bovine milk by added fatty acids (FAs) and monoglycerides (MGs) (Isaacs et al 1991) as well as by endogenous FAs and MGs (Isaacs et al 1986, 1990, 1991, 1992; Thormar et al 1987).

All three monoesters of lauric acid are shown to be active antimicrobials, i.e., alpha-, alpha'-, and beta-MG. Additionally, it is reported that the antimicrobial effects of the FAs and MGs are additive and total concentration is critical for inactivating viruses (Isaacs and Thormar 1990).

The properties that determine the anti-infective action of lipids are related to their structure; e.g., monoglycerides, free fatty acids. The monoglycerides are active, diglycerides and triglycerides are inactive. Of the saturated fatty acids, lauric acid has greater antiviral activity than either caprylic acid (C-10) or myristic acid (C-14).

The action attributed to monolaurin is that of solubilizing the lipids and phospholipids in the envelope of the virus causing the disintegration of the virus envelope. In effect, it is reported that the fatty acids and monoglycerides produce their killing/inactivating effect by lysing the (lipid bilayer) plasma membrane. However, there is evidence from recent studies that one antimicrobial effect is related to its interference with signal transduction (Projan et al 1994).

Some of the viruses inactivated by these lipids, in addition to HIV, are the measles virus, herpes simplex virus-1 (HSV-1), vesicular stomatitis virus (VSV), visna virus, and cytomegalovirus (CMV). Many of the pathogenic organisms reported to be inactivated by these antimicrobial lipids are those known to be responsible for opportunistic infections in HIV-positive individuals. For example, concurrent infection with cytomegalovirus is recognized as a serious complication for HIV+ individuals (Macallan et al 1993). Thus, it would appear to be important to investigate the practical aspects and the potential benefit of an adjunct nutritional support regimen for HIV-infected individuals, which will utilize those dietary fats that are sources of known anti-viral, anti-microbial, and anti-protozoal monoglycerides and fatty acids such as monolaurin and its precursor lauric acid.

No one in the mainstream nutrition community seems to have recognized the added potential of antimicrobial lipids in the treatment of HIV-infected or AIDS patients. These antimicrobial fatty acids and their derivatives are essentially non-toxic to man; they are produced in vivo by humans when they ingest those commonly available foods that contain adequate levels of medium-chain fatty acids such as lauric acid. According to the published research, lauric acid is one of the best "inactivating" fatty acids, and its monoglyceride is even more effective than the fatty acid alone (Kabara 1978, Sands et al 1978, Fletcher et al 1985, Kabara 1985).

The lipid coated (envelop) viruses are dependent on host lipids for their lipid constituents. The variability of fatty acids in the foods of individuals accounts for the variability of fatty acids in the virus envelop and also explains the variability of glycoprotein expression.

Loss of lauric acid from the American diet
Increasingly, over the past 40 years, the American diet has undergone major changes. Many of these changes involve changes of fats and oils. There has been an increasing supply of the partially hydrogenated trans-containing vegetable oils and a decreasing amount of the lauric acid-containing oils. As a result, there has been an increased consumption of trans fatty acids and linoleic acid and a decrease in the consumption of lauric acid. This type of change in diet has an effect on the fatty acids the body has available for metabolic activities.

VI. Lauric Acid in Foods
The coconut producing countries
Whole coconut as well as extracted coconut oil has been a mainstay in the food supply in many countries in parts of Asia and the Pacific Rim throughout the centuries. Recently though, there has been some replacement of coconut oil by other seed oils. This is unfortunate since the benefits gained from consuming an adequate amount of coconut oil are being lost.

Based on the per capita intake of coconut oil in 1985 as reported by Kaunitz (1992), the per capita daily intake of lauric acid can be approximated. For those major producing countries such as the Philippines, Indonesia, and Sri Lanka, and consuming countries such as Singapore, the daily intakes of lauric acid were approximately 7.3 grams (Philippines), 4.9 grams (Sri Lanka), 4.7 grams (Indonesia), and 2.8 grams (Singapore). In India, intake of lauric acid from coconut oil in the coconut growing areas (e.g., Kerala) range from about 12 to 20 grams per day (Eraly 1995), whereas the average for the rest of the country is less than half a gram. An average high of approximately 68 grams of lauric acid is calculated from the coconut oil intake previously reported by Prior et al (1981) for the Tokelau Islands. Other coconut producing countries may also have intakes of lauric acid in the same range.

The US experience
In the United States today, there is very little lauric acid in most of the foods. During the early part of the 20th Century and up until the late 1950s many people consumed heavy cream and high fat milk. These foods could have provided approximately 3 grams of lauric acid per day to many individuals. In addition, desiccated coconut was a popular food in homemade cakes, pies and cookies, as well as in commercial baked goods, and 1-2 tablespoons of desiccated coconut would have supplied 1-2 grams of lauric acid. Those foods made with the coconut oil based shortenings would have provided additional amounts.

Until two years ago, some of the commercially sold popcorn, at least in movie theaters, had coconut oil as the oil. This means that for those people lucky enough to consume this type of popcorn the possible lauric acid intake was 6 grams or more in a three (3) cup order.

Some infant formulas (but not all) have been good sources of lauric acid for infants. However, in the past 3-4 years there has been reformulation with a loss of a portion of coconut oil in these formulas, and a subsequent lowering of the lauric acid levels.

Only one US manufactured enteral formula contains lauric acid (e.g., Impact7); this is normally used in hospitals for tube feeding; it is reported to be very effective in reversing severe weight loss in AIDS patients, but it is discontinued when the patients leave the hospital because it is not sufficiently palatable for oral use. The more widely promoted enteral formulas (e.g., Ensure7, Nutren7) are not made with lauric oils, and, in fact, many are made with partially hydrogenated oils.

There are currently some candies sold in the US that are made with palm kernel oil, and a few specialty candies made with coconut oil and desiccated coconut. These can supply small amounts of lauric acid.

Cookies such as macaroons, if made with desiccated coconut, are good sources of lauric acid, supplying as much as 6 grams of lauric acid per macaroon (Red Mill). However, these cookies make up a small portion of the cookie market. Most cookies in the United States are no longer made with coconut oil shortenings; however, there was a time when many US cookies (e.g., Pepperidge Farm) were about 25% lauric acid.

Originally, one of the largest manufacturers of cream soups used coconut oil in the formulations. Many popular cracker manufacturers also used coconut oil as a spray coating. These products supplied a small amount of lauric acid on a daily basis for some people.

How much lauric acid is needed?
It is not known exactly how much food made with lauric oils is needed in order to have a protective level of lauric acid in the diet. Infants probably consume between 0.3 and 1 gram per kilogram of body weight if they are fed human milk or an enriched infant formula that contains coconut oil. This amount appears to have always been protective. Adults could probably benefit from the consumption of 10 to 20 grams of lauric acid per day. Growing children probably need about the same amounts as adults.

VII. Recommendations
The coconut oil industry needs to make the case for lauric acid now. It should not wait for the rapeseed industry to promote the argument for including lauric acid because of the increased demand for laurate. In fact lauric acid may prove to be a conditionally essential saturated fatty acid, and the research to establish this fact around the world needs to be vigorously promoted.

Although private sectors need to fight for their commodity through the offices of their trade associations, the various governments of coconut producing countries need to put pressure on WHO, FAO, and UNDP to recognizes the health importance of coconut oil and the other coconut products. Moreover, those representatives who are going to do the persuading need to believe that their message is scientifically correct -- because it is.

Among the critical foods and nutrition "buzz words" for the 21st Century is the term "functional foods." Clearly coconut oil fits the designation of a very important functional food.

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Kabara JJ. Inhibition of staphylococcus aureaus in The Pharmacological Effect of Lipids II (JJ Kabara, ed) American Oil Chemists' Society, Champaign IL, 1985, pp.71-75.

Kaunitz H, Dayrit CS. Coconut oil consumption and coronary heart disease. Philippine Journal of Internal Medicine 30:165-171;1992.

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Lim-Sylianco CY. Anticarcinogenic effect of coconut oil. The Philippine Journal of Coconut Studies 12:89-102;1987.

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Mendis S, Wissler RW, Bridenstine RT, Podbielski FJ. The effects of replacing coconut oil with corn oil on human serum lipid profiles and platelet derived factors active in atherogenesis. Nutrition Reports International 40:No.4;Oct.1989.

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About the Author

Mary G. Enig, PhD is an expert of international renown in the field of lipid biochemistry. She has headed a number of studies on the content and effects of trans fatty acids in America and Israel, and has successfully challenged government assertions that dietary animal fat causes cancer and heart disease. Recent scientific and media attention on the possible adverse health effects of trans fatty acids has brought increased attention to her work. She is a licensed nutritionist, certified by the Certification Board for Nutrition Specialists, a qualified expert witness, nutrition consultant to individuals, industry and state and federal governments, contributing editor to a number of scientific publications, Fellow of the American College of Nutrition and President of the Maryland Nutritionists Association. She is the author of over 60 technical papers and presentations, as well as a popular lecturer. Dr. Enig is currently working on the exploratory development of an adjunct therapy for AIDS using complete medium chain saturated fatty acids from whole foods. She is Vice-President of the Weston A Price Foundation and Scientific Editor of Wise Traditions as well as the author of Know Your Fats: The Complete Primer for Understanding the Nutrition of Fats, Oils, and Cholesterol, Bethesda Press, May 2000. She is the mother of three healthy children brought up on whole foods including butter, cream, eggs and meat.


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Monday, May 11, 2009

Cervical cancers after human papillomavirus vaccination

Cervical cancers after human papillomavirus vaccination.

Beller U, Abu-Rustum NR.

Shaare Zedek Medical Center, Jerusalem, Israel. beller@szmc.org.il

BACKGROUND: Current randomized clinical trials have shown that the quadrivalent human papillomavirus (HPV) vaccine can reduce the morbidity of precancerous lesions associated with HPV infection of vaccine-related subtypes. However, to date, there is no definite evidence showing the vaccine reduces the incidence of invasive cervical carcinoma. CASES: We present two cases--one young, vaccinated woman who developed cervical carcinoma that was unrelated to HPV and another who developed cervical carcinoma secondary to infection with an HPV subtype not covered by the vaccine. Both patients were treated successfully and remained well without evidence of cancer. CONCLUSION: Long-term follow-up data are needed to evaluate the prophylactic effectiveness of the current HPV vaccine. These cases could represent non-vaccine-related HPV infections. Young women must be thoroughly counseled about the efficacy and limitations of the vaccine and about continuing lifelong screening even after vaccination.

PMID: 19155953 [PubMed - in process]


Influence Of The Menstrual Cycle On The Female Brain

Influence Of The Menstrual Cycle On The Female Brain
ScienceDaily (Feb. 11, 2007) — What influence does the variation in estrogen level have on the activation of the female brain? Using functional Magnetic Resonance Imaging, Jean-Claude Dreher, a researcher at the Cognitive Neuroscience Center (CNRS/Université Lyon 1), in collaboration with an American team from the National Institute of Mental Health (Bethesda, Maryland) directed by Karen Berman, has identified, for the first time, the neural networks involved in processing reward-related functions modulated by female gonadal steroid hormones. This result, which was published online on January 29, 2007 on the PNAS website, is an important step in better comprehension of certain psychiatric and neurological pathologies.

See also:
Health & Medicine
Women's Health
Mind & Brain
Gender Difference
Functional neuroimaging
Dementia with Lewy bodies
The human brain has a recompense system that predicts different types of reward (food, money, drugs…). The normal functioning of this system plays a fundamental role in many cognitive processes such as motivation and learning. This reward system, composed of dopaminergic neurons(1) situated in the mesencephalon (a very deep region of the brain) and their projection sites(2), is crucial for neural coding of rewards. Its dysfunction can result in disorders such as addictions and is also implicated in various psychiatric and neurological pathologies, such as Parkinson's disease and schizophrenic disorders. Many studies on animals prove that the dopaminergic(3) system is sensitive to gonadal steroid hormones (estrogen, progesterone). For example, female rats self-administer cocaine (a drug that acts on the dopamine system) in higher doses after estrogens have been administered to them. The influence of gonadal steroid hormones on the activation of the reward system remained to be studied in humans. A better knowledge of this influence should make for better understanding of the differences between men and women, particularly as observed in the prevalence of certain psychiatric pathologies and in vulnerability to drugs, (for which the dopaminergic system plays an important role.) It is known, for example, that the female response to cocaine is greater in the follicular phase of the menstrual cycle(4) than in the luteal phase(5). Moreover, schizophrenia tends to appear later in women than in men.

Estrogens and progesterone are not just sex hormones that influence ovulation and reproduction; they also affect a large number of cognitive and affective functions.

These two observations show that gonadal neurosteroids(6) modulate the female dopaminergic system, but the question remains as to whether these hormones modulate the reward system neuron network.

In order to answer this question, the team developed an experiment using functional Magnetic Resonance Imaging (fMRI). The brain activity of a group of women was examined twice during their menstrual cycle. Each time they went into the MRI, they were presented with virtual slot machines showing different probabilities of winning. When women anticipate uncertain rewards, they activate the brain regions involved in processing emotions, particularly the amygdala and the orbitofrontal cortex, to a greater extent during the follicular phase (4 to 8 days after the start of the period) than during the luteal phase (6 to 10 after the LH(7) hormone surge). These results demonstrate increased reactivity of the female recompense system during the follicular phase, which is also the phase in which the estrogens do not oppose the progesterone. In order to determine the gender-related differences of reward system activation, the same experiment was carried out on a male group. Result: when men anticipate rewards, they mainly activate a region involved in motivation for obtaining rewards, the ventral striatum, whereas in women, it is a region dealing with emotions, the amygdalo-hippocampal region, which is the most highly activated.

These conclusions could be applied to rewards other than monetary. Take receptiveness and desire, for example, two qualities that are supposed to facilitate procreation and are seen during the period of ovulation. It could be envisaged that the increase in activity of certain regions of the female brain during the follicular phase would modulate behavior linked to obtaining rewards, such as approach behavior during reward anticipation and hedonistic behavior when the reward is received.

These results, at the border between neuroendocrinology and neurosciences, provide a better understanding of the fundamental role of gonadal steroid hormones on reward processing, particularly in behavioral processes such as motivation and learning. They also important in understanding the dysfunction of the reward system observed particularly in cases of Parkinson's disease, schizophrenia, normal ageing and drug and gambling addictions.

Adapted from materials provided by CNRS.

Herpesvirus, cytomegalovirus, human sperm and assisted fertilization

Herpesvirus, cytomegalovirus, human sperm and assisted fertilization
Coralie Pallier1, Lamia Tebourbi2, Stéphanie Chopineau-Proust1, Damien Schoevaert3, Patrice Nordmann1, Jacques Testart2 and Anne-Marie Courtot2,4
1 Service de Bactériologie–Virologie–Parasitologie–Hygiène, CHU Bicêtre, Paris-Sud, 78 rue du Général Leclerc, 94275 Le Kremlin–Bicêtre, 2 Institut National de la Santé et de la Recherche Médicale Unité 355 et Institut Fédératif de Recherche sur les Cytokines,32 rue des Carnets, 92140 Clamart and 3 Laboratoire d'Analyse d'Images en Pathologie Cellulaire, Institut Universitaire d'Hématologie, Hopital Saint Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France

Materials and methods

BACKGROUND: The effect of viral particles on the motility of human sperm and the relationship between sperm and virus are of importance particularly in assisted fertilizations. METHODS: We incubated ejaculated sperm with or without seminal fluid with either herpes simplex virus type 2 (HSV2) or human cytomegalovirus (HCMV). For each experiment, 5x105 sperm were incubated with a viral load of between 104 and 106 plaque-forming units. RESULTS: We detected no apparent variations in the percentage of motile forms when sperm were incubated with either HSV2 or HCMV. Using a computer-aided semen analysis system, a slight difference was reported in the percentage of motile forms when seminal fluid-free sperm were incubated with HSV2 (57.18 versus 64.43 in the control). Although the mean amplitude of lateral head displacement and the curvilinear velocity were significantly higher in infected sperm, the difference in straight line velocity was not statistically significantly different. Few viral particles (HSV2 or HCMV) adhered to the sperm membrane in the presence of seminal fluid. However, more particles stuck when in the absence of seminal fluid, particularly with HSV2 (8% of sperm sections for HSV2; 4% for HCMV). CONCLUSIONS: The relationship between sperm and viruses depends on the type of virus present as well as the presence or absence of seminal fluid. Motility is not a good enough criterion on which to prove the presence of viral elements, either in the medium or on the sperm.

Key words: cytomegalovirus/Herpesviridae/human sperm/motility/seminal fluid

Materials and methods

Several successful new assisted reproductive techniques have been developed over the last decade. It is now possible to take a selected spermatozoon from a given point in the genital tract and to transfer it into the ooplasm. This technique, known as ICSI, involves the use of micromanipulations to move sperm across the zona pellucida and the plasma membrane. The ICSI technique has enabled sperm that would otherwise be unable to fertilize the oocyte to do so, resulting in successful pregnancies. The sperm can be extracted from different regions of the genital tract, e.g. from the testes or from the epididymal duct (Lewin et al., 1999), or from semen samples. The extraction of testicular sperm followed by ICSI is a convenient way of overcoming non-obstructive azoospermia (Devroey et al., 1996). Several methods can be used to process the extracted sperm. The choice of method depends on the facilities available and the physiological condition of the sperm. These methods include isolating a simple swab in a synthetic medium (De Croo et al., 2000), washing by centrifugation (Devroey et al., 1996), selection on a Percoll gradient (Mercan et al., 2000) and swim-up migration (Marina et al., 1998). Although these techniques can solve many fertility problems, they also raise questions about the risks of transferring viral particles into the oocyte.

Several viruses are present in the genital tract. The most common are the herpes simplex virus type 2 (HSV2) and human cytomegalovirus (HCMV), which are sexually transmissible and can lead to fetal and neonatal anomalies (Huraux et al., 1999; Ranger-Roger et al., 1999). Sperm are formed in seminiferous tubules in the testes, where they are protected from the environmental tissue by the haematotesticular barrier. We previously showed that no viral particles are found within the seminiferous tubules in MCMV-infected murine testes. However, the peritubular cells, Leydig cells and endothelial cells contained high viral loads (Tebourbi et al., 2001). Several other papers have reported the presence of HSV2 (Deture et al., 1976) and of CMV (De Paepe et al., 1990) in the testes, but this does not automatically mean that the seminiferous tubules were contaminated (Baskar et al., 1986; Stephanopoulos et al., 1989). However, viruses may be present in the epididymis (Dalton and Harcourt-Webster, 1991; MacCarthy et al., 1991), deferent duct (Kimura et al., 1993), prostate (Boldogh et al., 1983; Benson and Smith, 1992; Mastroianni et al., 1996) or seminal vesicles (Deture et al., 1976; Kimura et al., 1993). If the virus is present in the annexial glands, it may be discharged in the seminal fluid before coming into contact with sperm. The presence of the virus in the semen indicates a genital tract infection. One study (Moore et al., 1989) found infective HSV2 virus in the semen of a healthy donor with an asymptomatic primary infection, and showed that insemination led to the horizontal transmission of the virus. However, cell culture methods have rarely detected HSV2 in sperm (Deture et al., 1978; MacGowan et al., 1983), because semen contains inhibitors that may interfere with these methods (Deture et al., 1978; Sherman and Morgan, 1989). More sensitive methods, such as PCR and in-situ hybridization (ISH), have made it possible to detect viral DNA in the semen of men with genital HSV2 infection (Kotronias and Kapranos, 1998; Wald et al., 1999) and in the semen of a high-risk population (Tabrizi et al., 2000). HCMV has been identified in sperm (Lang and Kummer, 1972). The prevalence of HCMV elements was found to be variable in men who did not have human immunodeficiency virus (HIV) or other sexually transmitted diseases (Lang and Kummer, 1972; Bantel-Schaal et al., 1993; Shen et al., 1994; Mansat et al., 1997), but it may be high in high-risk populations (Biggar et al., 1983; Leach et al., 1993). Detection rates are generally higher for viral DNA than for infective particles. In a recent study involving men taking part in an IVF programme (Witz et al., 1999), 25% of ejaculates, including those from HCMV-negative men (who may or may not have gone on to become positive), were found to contain viral DNA.

Thus, sperm are protected from viral contact in the upper regions of the genital tract, i.e. the testes, whereas sperm in the semen can easily come into contact with viral particles (in the epididymis, deferent duct and annexial glands). This is important as sperm can be harvested from testicular biopsies for ICSI in cases of azoospermia. This then disrupts the haematotesticular barrier, thus removing the protective effect and placing the sperm in close contact with endothelial cells, Leydig cells and peritubular cells. Sperm can also be harvested from the other genital organs or from the semen. In this case, the fertilizing sperm for ICSI are choosen according to their motility. The relationship between the virus and sperm motility is not very clear and the interaction between sperm and virus are of importance.

Some authors have reported an inverse correlation between the presence of HSV2 in semen and sperm motility (Kotronias and Kapranos, 1998). Similar observations have been reported concerning CMV (Torino et al., 1987). A high concentration of HCMV was detected in the semen of a patient with CMV mononucleosis, and this was associated with a transient decrease in sperm motility (Lang and Kummer, 1972; Lang et al., 1974). We were interested to know whether this decrease in sperm motility was due to a direct effect of high concentrations of the virus on sperm or if these results were due to an indirect effect. Few papers have addressed the viral load of semen (Lang and Kummer, 1972; Biggar et al., 1983). Therefore it is not clear how the virus and sperm interact. No relationship has been found between sperm and HSV2 (Sherman and Morgan, 1989), whereas ISH has been used to demonstrate that there is a close relationship between sperm and HSV2 (Kotronias and Kapranos, 1998). Only one group has found CMV within human sperm (Huang et al., 1986); another group (Baskar et al., 1986) observed virus-like particles in mouse sperm.

We developed experimental models to analyse the effects of viruses on sperm motility and the interaction between viruses and sperm in assisted reproduction indications. On the one hand, we used semen, i.e. sperm plus seminal fluid, which is the physiological environment from which motile sperm are selected for intrauterine insemination, IVF and ICSI. On the other hand, we used sperm without seminal fluid (selected by use of a PureSperm gradient), which corresponds to sperm collected in the epididymis before the formation of seminal fluid or to sperm collected from testicular biopsies in azoospermic men for ICSI. The viruses used were HSV2 and HCMV. This study enabled us to study the relationship between viruses and sperm in different regions of the genital tract and to determine whether the new assisted reproduction techniques (especially ICSI in the case of azoospermia) affect the risks of virus adherence to sperm, leading to the introduction of viral particles into oocytes.

Materials and methods
Materials and methods

Preparation of the viral strains
The viral strains, HSV2 (ATCC VR-2019) and HCMV (AD-169), were prepared from human embryo lung cells (MRC5, primary cells; Biomerieux, Lyon, France) in modified Eagle's medium (MEM; Gibco BRL, Life Technologies, Paisley, UK). The viral suspensions were titrated by an enzymatic assay method (visualization of early viral antigens) at a concentration of 2x107 plaque-forming units (PFU)/ml.

Human sperm
Sperm samples were taken from male patients undergoing IVF or who attended the American Hospital in Neuilly (France) with fertility problems. The selected patients all had 50% motile sperm, as tested in seminal fluid (six samples) or after selection on PureSperm (JOD, Lyon, France) gradient (five samples). For PureSperm gradient selection, 0.2–2 ml of semen was passed over two successive layers of PureSperm. The culture medium was used to prepare a 45% dilution of the upper layer and a 90% dilution of the 1 ml lower layer. After centrifugation (600 g for 20 min), the sperm pellet was washed in 10 ml PBS (600 g for 10 min) and then diluted in MEM.

Sperm incubated with virus
Sperm (5x105 sperm in 125 µl of MEM) were incubated with the virus (<104 to 106 PFU for 105 sperm) in a Petri dish (Corning 35 mm, tissue culture) for 1 h at 37°C in a moist atmosphere containing 5% CO2. In the controls, the viral suspension was replaced by the culture medium used to prepare the cells.

Interaction between sperm and virus
Sperm motility
Analysis at the optical level. After 1 h incubation, 50 µl of each sample was placed on a slide and the percentage of motile sperm was assessed under the microscope by three independent observers.

Analysis with a CASA system. We used a CASA system to analyse sperm motility. The system used was composed of a microscope (Olympus BHx200), a high speed video camera (Lhesa-Electronic) with a video recorder (Sony time lapse 168) associated with a sytem consisting of a pentium PC, a digitalized Matrox PIP card and an original Siam-Trac software. The following parameters were studied: percentage of motile sperm, straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN) mean amplitude of lateral head displacement (ALH) and linear acceleration (LA).

For this analysis, sperm were selected on PureSperm gradient and incubated with HSV2 for 1 h (106 sperm/106 PFU in 125 µl DMEM). The control was treated in the same way but replacing the viral suspension with culture medium.

Ultrastructural analysis
The sperm were then incubated with the viral suspensions for 1 h in a glutaraldehyde solution (2.5% in Sörensen buffer). After washing several times with the same buffer to which 1 mol/l sucrose solution had been added, the sperm were incubated in osmic acid (1% in Sörensen buffer) for 1 h before dehydrating by successive passages in 70°, 90°, 95° (3x10 min) and 100° (3x20 min) ethanol. This was followed by a final incubation in araldite for 3 days at 60°C. Ultrathin sections were recovered on 600 mesh grids, before being stained with uranyl acetate in ethanol and then with lead citrate. The sections were examined under a Hitachi electron microscope at a magnification of x12 000. The sections analysed were 10 µm away from each other. The proportion of sperm sections (95 nm thickness) in which viral particles were attached to the membrane or contained in the cell was determined for each experimental condition.

Paired t-tests were used to compare sperm motility (CASA) parameters of seminal fluid-free sperm incubated with either HSV2 or with culture medium. P 0.05 was considered significant.

Materials and methods

Sperm motility
Analysis at the optical level
The percentage of motile sperm was evaluated after incubation with HSV2 (seven samples) or with HCMV (nine samples). Both viruses were tested on samples consisting of sperm plus seminal fluid (HSV2: three samples; HCMV: four samples), and samples of sperm separated from the seminal fluid on PureSperm gradient selection (HSV2: four samples; HCMV: five samples). Several viral concentrations were used (<104, 105 and 106 PFU). Table I summarizes the results obtained with seminal fluid-free sperm incubated with HSV2 culture medium. The percentage of motile sperm in the experimental and control samples did not differ significantly, regardless of the procedure, viral strain or viral concentration used.

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Table I. Comparison of the percentage of motile sperm between seminal fluid-free sperm incubated with HSV2 and with culture medium by optical analysis

Analysis with a CASA system
Table II summarizes the analysis of sperm motility parameters (CASA analysis) of seminal fluid-free sperm incubated with HSV2 or with culture medium alone. A slight but insignificant decrease in the percentage motile sperm was reported when sperm were incubated with HSV2 (57.18 versus 64.43% for control). Although VSL was not different in the two groups (22.97 versus 25.66 µm/s for control), VCL differed (82.90 versus 67.23 µm/s for control; P = 0.05), lowering LIN in the experimental group (0.27 versus 0.38% for control; P = 0.05). ALH was significantly higher when sperm were in the presence of HSV2 (3.48 versus 2.46 µm for control; P = 0.05) as was the linear acceleration (LA; 608.62 versus 474.84 m/s2; P = 0.05).

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Table II. Comparison of the sperm motility parameters with computer-assisted semen analysis between seminal fluid-free sperm incubated with HSV2 and with culture medium

Ultrastructural analysis
Sperm with seminal fluid incubated with HSV2 or HCMV
HSV2. Two samples were subjected to ultrastructural analysis and viral particles were detected in both. The particles observed were in the form of complete virions and isolated capsids, but they were seldom found in contact with the sperm membrane (two particles in 310 sperm (0.6%) for sample 1, none in 154 sperm for sample 2).
HCMV. Two samples underwent ultrastructural analysis. Complete and incomplete viral particles, and dense bodies were found to co-exist. Only a few of the sperm were in close contact with viral particles [two out of 238 sperm for sample 1 (0.8 %), and two out of 319 sperm for sample 2 (0.6 %)].

Sperm without seminal fluid incubated with HSV2 or HCMV
A comparative study was carried out using sperm obtained from the same ejaculate and selected by PureSperm gradient. They were incubated with either HSV2 or HCMV.

HSV2. We found 28 viral particles in close contact with the 350 sperm sections observed (8%). The viral particles were either very near the membrane or directly stuck to the membrane of the sperm head (Figure 1E could correspond to fusion of HSV2 virion with the sperm membrane) or of the flagella (Figure 1B,C,D). We did not detect any viral particles inside the sperm. Viral particles were also observed either in isolation or in clusters in the preparation (Figure 1A), showing that they had not been removed from the sperm sample exposed to HSV2 despite the washes carried out before inclusion in araldite.

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Figure 1. (A) Herpes simplex virus 2 (HSV2) viral particles in the sample. The viruses were present in the form of the complete virion with one or several capsids, or in incomplete forms with a full or empty capsid and thick electron-dense coat. Bar = 500 nm. (B, C) Two sperm with viral particles (arrows) attached to the membrane of the principal piece. Bar = 200 nm. (D) HSV2 viral particle (arrow) attached tothe membrane of the intermediate piece. Bar = 500 nm. (E) Viral particles (arrows) attached to the sperm membrane of the acrosome. Bar = 500 nm.

HCMV. Isolated and clustered viral particles were also observed in the sperm sample incubated with HCMV (Figure 2A). We observed viral particles close to the membrane (Figure 2B) in 15 of the 381 sperm sections analysed (4%), implying that the relationship between the sperm and viral particles may be weaker for HCMV than for HSV2.

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Figure 2. (A) Human cytomegalovirus particles. The viruses were present in the form of complete virions with one capsid, or inincomplete forms with empty isolated capsids, or dense bodies. Bar = 500 nm. (B) Dense body (arrow) attached to the sperm membrane. Bar = 500 nm.

Materials and methods

We have clearly shown that: (i) high concentrations of HSV2 or HCMV do not have a strong effect on the percentage of motile sperm regardless of the viral concentration used or the method used to prepare the sperm; however, motility parameters detected by CASA differed when HSV2 was added to seminal fluid-free sperm; (ii) there was little interaction between HSV2 or HCMV and human sperm when the viruses were incubated with the semen (sperm plus seminal fluid); HSV2 and HCMV behaved differently when incubated with sperm in the absence of seminal fluid; more HSV2 adhered to sperm than HCMV.
Some studies have shown that the presence of viral particles in the semen is correlated with a lower percentage of motile sperm (Lai et al., 1997; Kotronias and Kapranos, 1998). Our study shows that the presence of HSV2 or HCMV in the sperm suspension does not inhibit sperm motility, regardless of the viral concentrations used and the method used to treat the sperm: sperm with seminal fluid, sperm selected by PureSperm gradient (no seminal fluid), or sperm selected by centrifugation and migration (data not shown). These observations were the same for all the samples studied, and CASA system applied to HSV2 strengthened our results. It would therefore appear that the in-vivo correlation between the reported reduction of percentage of motile sperm and the presence of viral particles (Lai et al., 1997; Kotronias and Kapranos, 1998) is due to indirect interactions between the virus and the sperm rather than to a direct effect on motility. However, we observed modifications of motility parameters such as VCL, LIN, ALH and LA, showing that in the presence of HSV2, sperm moved more rapidly with a greater ALH.

We showed that human sperm do not bind to HSV2 in the presence of seminal fluid, although free viral particles were found in the seminal fluid. These findings are similar to those of other investigators (Sherman and Morgan, 1989) who did not find a close relationship between sperm and HSV2, and those of another study (Deture et al., 1978), which observed free viral particles in the seminal fluid. The seminal fluid seems to impede the adherence of HSV2 with sperm. Sherman and Morgan (1989) hypothesized that spermine phosphate is involved. Seminal fluid contains high molecular weight proteins that could impede the interaction of HSV2 with sperm or behave as a receptor for HSV2 thus preventing further contact by sperm.

Conversely, in the absence of seminal fluid, sperm were able to adhere to HSV2. It was difficult to evaluate the exact percentage of sperm with HSV2 adhering to their surface because we observed sections of sperm (95 nm thick) and not whole sperm (4–5 µm thick). The percentage of sperm bearing viral particles was 8% but may have been as high as 45% if only one viral particle adhered to each sperm. This percentage is within the range described for ISH HSV2 DNA-positive sperm cells (Kotronias and Kapranos, 1998). These results also raised questions about the nature of the relationship between HSV2 and sperm. It may consist of a simple passive attachment that is able to resist the repeated washes. This is highly possible given our results. However, in one spermatozoon, the virus particle seemed to be closer to the sperm membrane, and this could even be a beginning of fusion. Thus, we cannot exclude the possibility that this is a true ligand/receptor relationship that only occurs when sperm have been separated from their seminal fluid. Numerous HSV2 receptors have been identified, including heparan sulphate-like glycosaminoglycans (Trybala et al., 2000), nectins and a receptor belonging to the tumour-necrosis factor family (Campadelli-Fiume et al., 2000). It is possible that the sperm have one of these membrane receptors, which is not accessible to the virus when seminal fluid is present.

In conclusion, sperm motility, which is the parameter used to select sperm for ICSI, is not a good enough criterion on which to prove the presence of viral elements in the medium or on the sperm.

From our experiments with virus and sperm with seminal fluid, we can conclude that when the virus is mixed with semen in the annexial glands in vivo, HSV2 does not interact with the sperm and the virus remains in the seminal fluid. In these conditions, the techniques used in assisted reproduction to eliminate the seminal fluids also eliminate the viral particles and improve the sperm preparation. This means that either PureSperm gradient selection or swim-up migration can be used efficiently.

Sperm inside the seminiferous tubules are protected from viruses by the haematotesticular barrier. When this barrier is ruptured, sperm can come into contact with viral particles. Indeed, this barrier is always destroyed when sperm are collected from the testis. This increases the risk of sperm coming into contact with high local concentrations of HSV2 due to contamination from the blood or interstitial tissues. The washing procedure cannot eliminate viral particles, meaning that they are directly injected into the cytoplasm.

The HCMV virus particle did not interact with the sperm when HCMV was added to the semen. However, the relationship between sperm and virus in the absence of the seminal fluid was not so clear as for HSV2, perhaps due to the fact that this virus binds non-specifically. However, as for HSV2, the viral particle may be accidentally injected into the ooplasm during ICSI, either as a result of passive transport by the sperm or from the surrounding environment as washing did not eliminate the virus from the preparation.

Some studies used sperm as vectors (Perry et al., 1999). These experiments concerned purified DNA or plasmids, and we did not study this aspect of the problem as we focused only on infectious virions visible under the electron microscope.

We have attempted to investigate whether there may be a specific risk of viral infection associated with ICSI. Our main conclusion is that ICSI creates a new risk of contaminating sperm with viruses in cases of ICSI for azoospermia. Sperm inside the seminiferous tubules are protected from viruses by the haematotesticular barrier. When this barrier is ruptured, sperm can come into contact with viral particles. Indeed, this barrier is always destroyed when sperm are collected from the testes. This increases the risk of sperm coming into contact with high local concentrations of HSV2 due to contamination from the blood or interstitial tissues. The washing procedure cannot eliminate viral particles, meaning that they are directly injected into the ooplasm.

Materials and methods

We would like to thank Pascal Briaud, from the American Hospital, Neuilly and Claudine Hemon from INSERM U355, Clamart, for providing us with sperm samples for this study and Phillipe Etre for the electron micrographs. Electron microscopic observations were carried out in the Neurology Laboratory of Professor Gerard Said (CHU Bicêtre, Paris Sud) with technical help from Josette Bacci.


4 To whom correspondence should be addressed. E-mail: anne-marie.courtot@inserm.ipsc.u-psud.fr

Materials and methods

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Submitted on October 8, 2001; accepted on January 2, 2002.



Expert Interview: Meet Francis L. Hutchins, M.D.

If there were such a thing as a "fibroid king", Francis L. Hutchins, M.D. could rightfully wear that crown. A renown fibroid expert, Dr. Hutchins has developed a practice that is devoted almost entirely to treating women with fibroids. Most recently, Dr. Hutchins has been recognized in the media for his pioneering efforts in using uterine artery embolization to treat women with severe bleeding due to fibroids. A Clinical Associate Professor of Obstetrics and Gynecology at Allegheny University of the Health Sciences and Thomas Jefferson University, Dr. Hutchins is passionate when it comes to educating women about fibroids and helping them sort out the best treatment option for their particular situation. You may want to consult his website Hope For Fibroids Organization for additional fibroid information. Here’s what he had to say about fibroids during a recent interview.

When does a woman with fibroids require at hysterectomy?

Dr. Hutchins: Most women with fibroids do not need to have a hysterectomy. And I believe that we are going to see the numbers of hysterectomies performed to treat fibroid symptoms decline, as more doctors and women become aware of other effective, less-invasive treatment options. A hysterectomy to treat fibroids should be reserved for the woman who says, I’ve been burned. I’ve tried everything and I don’t want to ever have to think about my fibroids again. I want cure! If you want cure, then removal of the uterus is the answer to your problem. But, deciding to undergo a hysterectomy is a big decision and a woman needs to understand the risks associated with the surgery and other serious issues that go along with it. I only consider this when all else has failed.

How does a woman know her doctor is up to date and qualified to perform the newer techniques available to treat fibroid symptoms?

Dr. Hutchins: I think that especially because there has been a rapid expansion of treatment options in the past ten years, that it is more incumbent than usual for the woman to do her homework as best as possible get a grasp on what a full range of options are and expect that her gynecologist will be able to discuss those within the framework of what her needs are. And the woman should be able to make liberal use of second and third opinions. If someone says they can do something, that is materially different than if they say they do this particular thing. If a gynecologist is not performing myomectomies multiple times a year, then that individual is probably not skilled in that procedure and is not necessarily the best person to be performing it, especially if fertility is the issue. So, the question that should be put to the gynecologist is, How regularly do you perform this or that procedure?

Does a woman with fibroid have to worry about it turning cancerous?

Dr. Hutchins: The consensus opinion today is that a fibroid is a fibroid is a fibroid. It is absolutely unnecessary for a woman to be concerned whether or not that fibroid is a sarcoma, an extremely rare cancer that can look like a fibroid. Years ago, when I was in medical school, we were taught that a fibroid can turn into a sarcoma, but no one believes that today.

What is the greatest myth that women have about fibroids?

Dr. Hutchins: Number one is that they have to be treated at all and number two is that they are going to eventually have symptoms and require a hysterectomy.

What do women fear the most about their fibroids?

Dr. Hutchins: Most women fear they are going to need a hysterectomy. And the interesting thing about that is that many times they are criticized by their female friends for not just going on and being a woman about it. There’s that mentality: have your hysterectomy like everyone else. What’s the matter with you, you punk! I meet women all the time who are burdened with this guilt, because they are "too chicken" to have a hysterectomy. I want to change this way of thinking.


This article courtesy of EmpowerMed, an Internet-based service that empowers individuals and their families to become more active participants in the decisions that affect their health and well-being. This free service will be available in April, 1998 at www.empowermed.com. Copyright 1998 EmpowerMed, all rights reserved.

Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure

Systemic lupus erythematosus in adults is associated with previous Epstein-Barr virus exposure
Judith A. James 1 *, Barbara R. Neas 1, Kathy L. Moser 2, Teresa Hall 3, Gail R. Bruner 3, Andrea L. Sestak 3, John B. Harley 4
1University of Oklahoma Health Sciences Center, Oklahoma City
2Case Western Reserve University, Cleveland, Ohio
3Oklahoma Medical Research Foundation, Oklahoma City
4University of Oklahoma Health Sciences Center and US Department of Veterans Affairs, Oklahoma City, Oklahoma

*Correspondence to Judith A. James, Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104

Funded by:
NIH; Grant Number: AR-42474, AR-01981, AI-31584, AR-45084, AI-24717, AR-42460, AR-45231, N01-AR-52221
US Department of Veterans Affairs


The possible molecular mimicry of the Epstein-Barr virus (EBV) peptide PPPGRRP by the peptide PPPGMRPP from Sm B/B of the human spliceosome is consistent with the possibility that EBV infection is related to the origin of systemic lupus erythematosus (SLE) in some patients. Association of EBV exposure with SLE was therefore tested for and subsequently found in children and adolescents (odds ratio [OR] 49.9, 95% confidence interval [95% CI] 9.3-1,025, P < 10-11). These results were confirmed at the level of EBV DNA (OR > 10, 95% CI 2.53-, P < 0.002). Much smaller seroconversion rate differences were found against 4 other herpes viruses. Herein, we extend these studies to adults and test the hypothesis that EBV infection is associated with adult SLE.

We selected 196 antinuclear antibody-positive adult SLE patients (age 20 years) and 2 age-, race-, and sex-matched controls per patient. SLE patients and matched controls were tested for evidence of previous infection with EBV, cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), or varicella-zoster virus (VZV) by standardized enzyme-linked immunosorbent assays.

Of the 196 lupus patients tested, all but 1 had been exposed to EBV, while 22 of the 392 controls did not have antibodies consistent with previous EBV exposure (OR 9.35, 95% CI 1.45-, P = 0.014). No differences were observed between SLE patients and controls in the seroconversion rate against CMV, HSV-2, or VZV.

These new data from adults, along with the many suggestive features of EBV infection, are consistent with the contribution of this infection to the etiology of SLE.

Received: 15 February 2000; Accepted: 27 September 2000
Digital Object Identifier (DOI)

10.1002/1529-0131(200105)44:5<1122::AID-ANR193>3.0.CO;2-D About DOI

Article Text

Systemic lupus erythematosus (SLE) is an idiopathic, autoimmune disorder in which autoantibodies are universally present. Unfortunately, the mechanisms leading to the production and perpetuation of these aberrant autoimmune responses remain poorly understood. Autoantibodies directed against the spliceosomal proteins, anti-Sm and anti-nuclear RNP, are found in 30-50% of lupus patients' sera ([1]). The Sm B/B protein is a major target of this autoimmune response. Epitope mapping in our laboratory has identified the specific regions of this protein that are commonly targeted by lupus patient sera ([2]). Among the B cell epitopes defined by overlapping octapeptides ([2]), PPPGMRPP consistently appears earliest in the anti-Sm autoimmune response of human lupus ([3]).

Immunization with PPPGMRPP induces lupus autoimmunity ([4-6]). A structurally similar and antigenically crossreacting peptide from Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) also induces lupus humoral autoimmunity ([7]). These results, as well as conflicting reports in the literature, led us to test the hypothesis that EBV is associated with SLE. Seroconversion against EBV failed to occur in only 1 of 117 pediatric lupus patients compared with 46 of 153 controls (odds ratio [OR] 49.9, 95% confidence interval [95% CI] 9.3-1,025, P < 10-11) ([8]). Similar results have been obtained confirming the association of serologic evidence of EBV infection with SLE in children and adolescents ([9]). In both studies ([8][9]), much smaller seroconversion rate differences were found against 4 other herpes viruses: cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV). EBV infection was identified by polymerase chain reaction for EBV DNA in 32 of 32 lupus patients (100%) and in 23 of 32 matched controls (72%). In all 9 of the discordant sets, EBV DNA was found in the lupus patient, but not in the matched control (OR >10, 95% CI 2.53-, P < 0.002) ([8]). These data, along with the many suggestive features of this viral infection, are consistent with the immune response against EBV infection being a potential triggering mechanism for SLE.

Initially, pediatric and adolescent lupus patients were selected because of the lower frequency of previous EBV exposure in these normal populations ([10]). Although pediatric and adult SLE are very similar and even share the same diagnostic criteria ([11][12]), the etiologies of SLE are not necessarily the same in younger and older SLE patients. To determine whether the association of SLE with EBV infection observed in children and adolescents ([8]) could be extended to adults, we selected a group of 196 adult lupus patients and 392 normal controls and evaluated their exposure to EBV.


Patients and controls

We studied antinuclear antibody-positive SLE patients who satisfied the classification criteria of the American College of Rheumatology ([11][12]). All patients were 20 years of age at the time of specimen collection. Patients were recruited from pedigrees of genetic studies being conducted in our laboratory. Virtually all of the SLE patients were members of multiplex lupus families. Controls were matched by age (±10 years), race (self declared), and sex. We selected 196 adult lupus patients and 392 adult controls from 169 pedigrees in our lupus genetics study. Of the 169 pedigrees, 108 included an SLE patient. We also organized age into 10-year intervals. Serum samples had been stored at -20°C since collection.

Anti-Epstein Barr virus viral capsid antigen (anti-EBV-VCA) antibodies

Evidence of previous EBV infection was detected by measuring IgG antibodies against purified EBV-VCA (Wampole, Cranbury, NJ), following the manufacturer's instructions. The data analysis used the international standardized ratio (ISR), a semiquantitative measure of the relative level of antibody that is standardized between assays by known, characterized, positive controls. The ISR is designed to optimally detect seroconversion.

Occasionally, serum specimens produced an equivocal result (ISR 0.9-1.10), and these samples were repeat-tested. To avoid falsely identifying any serum sample as showing no evidence of seroconversion, we uniformly reported as positive any serum sample showing 2 equivocal readings as positive.

Antiviral assays

Serum specimens were also tested for reactivity with CMV, HSV-1, HSV-2, and VZV. Again, these specimens were tested and ISRs calculated by a standardized enzyme-linked immunosorbent assay (Wampole), following the manufacturer's instructions.

Statistical analysis

The statistical analysis adjusted for the correlated nature of the family data and accounted for the potential confounder of age. The relationship between seroconversion and patient status was assessed using conditional logistic regression with pedigree as the matching factor. The models were adjusted for age by including the variable representing 10-year age intervals. Due to the small number of individuals who were not seropositive for some of the viruses studied, all analyses were performed using LogXact 2.1 ([13]). The results presented are exact P values and 95% CIs. The ISR comparisons were analyzed using a mixed fixed-effects and random-effects model (PROC MIXED; SAS Institute, Cary, NC) to account for the pedigree effect. We also calculated least-squares (LS) means for all mean comparisons.


We studied 196 SLE patients and 392 unaffected controls from a total of 169 pedigrees. No controls reported having SLE, and we adjusted all analyses for age. The patients and controls were 66% European American, 30% African American, and 4% other; 94% were women. Demographics of the patients and controls are presented in Table 1. All patients and controls were recruited from families that were multiplex for lupus. (A separate study will be needed to test whether an association exists between EBV infection and the nongenetic or sporadic instances of SLE in simplex pedigrees. No study has yet shown, however, that SLE in multiplex pedigrees is fundamentally different from that in simplex lupus pedigrees.)

Table 1. Demographics of adult lupus patients and matched controls


Lupus patients Normal controls


Age, mean ± SD years 44.7 ± 12.4 45.9 ± 12.9
Age range, years 20-76 20-84
Women, % 94 94
No. of families represented 108 151
Race, %
Caucasian 65 66
African American 30 30
Other* 5 4


* Participants identifying themselves as predominantly of Hispanic, American Indian, Asian, or Eastern Indian heritage.

Sera from each patient and control were tested for evidence of previous EBV infection. If there was sufficient binding of IgG directed against EBV-VCA, the subject was said to have seroconverted, although a conversion from negative to positive is not ordinarily observed. Of the 196 adult lupus patients evaluated, 195 (99.5%) had been previously exposed to EBV by this criterion, while a smaller proportion of controls (370 of 392, or 94.4%) showed evidence of seroconversion (OR 9.35, P = 0.014) (Table 2).

Table 2. Seroconversion against Epstein-Barr virus viral capsid antigen (EBV-VCA), cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV) in sera from adult lupus patients and controls*


Lupus patients, no. positive/total tested (%) Normal controls, no. positive/ total tested (%) OR 95% CI P


EBV-VCA 195/196 (99.5) 370/392 (94.4) 9.35 1.45- 0.014
CMV 130/196 (66.3) 270/392 (68.9) 0.97 0.56-1.70 1.0
HSV-1 161/196 (82.1) 311/392 (79.3) 1.32 1.02-1.86 0.03
HSV-2 123/196 (62.8) 217/392 (55.4) 1.11 0.68-1.84 0.74
VZV 193/195 (99.0) 385/392 (98.2) - - 0.56


* Sera from lupus patients or their controls were tested for IgG antibodies against EBV-VCA, CMV, HSV-1, HSV-2, and VZV, and were standardized for seroconversion. Ninety-five percent confidence intervals (95% CI) and P values are exact values obtained from the LogXact 2.1 program ([13]). OR = odds ratio.
There were too few discordant values to provide valid estimates of the OR and 95% CI.

In addition, as shown in Figure 1, SLE patients had a higher titer of anti-EBV-VCA antibodies compared with unaffected controls (LS mean patient ISR 4.18, LS mean control ISR 2.59; t = 10.65, P < 4 × 10-19) when we accounted for the familial clustering. None of the remaining responses had so large a difference in the effect, although the LS means were significantly different for CMV (LS mean patient ISR 2.20, LS mean control ISR 1.72; P < 2 × 10-4) and for VZV (LS mean patient ISR 3.11, LS mean control ISR 2.72; P < 1 × 10-5). Neither HSV-1 nor HSV-2 showed a significant increase in the SLE patients. The ISR value difference for EBV was 3.3-fold that for CMV and 4.1-fold that for VZV.

Figure 1. International standardized ratios (ISRs) for reactivity of lupus patient and normal control sera with Epstein-Barr virus (EBV) viral capsid antigen, cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV). Mean ISRs for each antigen by patient and control group are presented as heavy horizontal lines, and confidence intervals are presented as thin vertical lines above and below these means.
[Normal View 39K | Magnified View 56K]

Of the antibodies to the herpes viruses measured in this study, those against EBV showed the strongest statistical and practical association with SLE when we accounted for familial clustering and controlled for age. Although HSV-1 produced a statistically significant result, the OR was only 1.32.


EBV has been known for more than 4 decades. Why has the association of EBV infection with SLE been so difficult to establish? First, the near ubiquity of EBV infection among adults means that if EBV contributes to the etiology of SLE, then SLE is an uncommon consequence of EBV infection and there must be another explanation for the sporadic occurrence of SLE. The likely reasons have to do with EBV itself (e.g., risk of infection varying with EBV strain), the circumstances of EBV exposure (i.e., route, dose, concurrent infection, age of host), the host immune response (e.g., stochastic processes), and/or the genetics of the host. Without pertinent data from testing these possibilities, however, we can only speculate about the reasons that we consistently observe an association of EBV exposure with SLE. In some ways, these considerations are not relevant to the validity of the observed association, although they may, of course, form the basis for exploring a possible role of EBV in the etiology of SLE.

Second, studies performed before the 1997 report of an association ([8]) all relied upon assays that were <90% sensitive for the presence of EBV infection. When the expected true seronegative rate in adults is 5%, even a false-negative rate of only 5% in detecting seroconversion against EBV will wreak havoc with the power of a study to detect an effect that is present. This means that studies that showed a possible association ([14-16]), as well as those that did not ([17-19]), contained too few SLE patients and controls to reliably and reproducibly detect a difference. In addition, only one of the previous studies used the strategy of evaluating children and adolescents ([19]), which would increase the ability to detect associations. However, this study was also underpowered and used an anti-EBV antibody assay and EBV DNA assay technologies that were even older and less reliable.

Four studies have previously evaluated the role of CMV in SLE ([8][9][19][20]). Three of them found no association of CMV seroconversion with SLE ([8][9][19]), although one study was performed in a small set of pediatric patients ([19]). The two other studies evaluated a combined total of 143 children and adolescents with SLE, along with a larger control group. A fourth study showed an association in a sample of 97 adult SLE patients compared with 97 controls ([20]). Controls were not matched by ethnic background, geographic location, or socioeconomic status. These may be important variables to control for and could certainly explain the differences these investigators observed. In addition, the prevalence of anti-CMV antibodies in the control subjects (43%) was lower than (and outside the range of) that in previous seroepidemiologic studies (54% to >90%). Therefore, variation in assays may also account for some of the difference and explain why those investigators found an association of SLE with serologic evidence of CMV infection ([20]), while we and others did not ([8][9][19]).

EBV is an attractive component of the environmental risk for SLE for other reasons. First, EBV infection is found worldwide and spares no known segment of the human population, which is similar to the distribution of SLE. Second, after infection, EBV remains viable and actively infects the host for life. Third, EBV infection is a continuous source of chronic immune stimulation. Indeed, the latent form of EBV actually infects B cells and is perpetuated in them. These same cells are critical for the production and propagation of autoantibodies, which are central to the laboratory diagnosis of SLE as well as to many of its clinical manifestations. Fourth, EBV periodically reactivates, which could hypothetically be associated with increased production of lupus autoantibodies and perhaps disease flares. Finally, EBNA-1 (the protein with the PPPGRRP and GRGRGRGR sequences that have significant homology with lupus autoantigens) is expressed in latently infected B cells. However, the ubiquitous nature of EBV suggests that if infection with this virus is necessary to the development of lupus, then there must be something about the usual human host's immune response that prevents lupus from becoming a much more widespread disease.


We would like to thank the Lupus Multiplex Registry and Repository (supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases grant AR-52221) for the use of their well-characterized lupus patient and control materials. We would also like to thank Jama Kendall, Amber Davis, and Xana Kim for their technical assistance.


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Firman Abdullah Bung

drFirman Abdullah SpOG / ObGyn

drFirman Abdullah SpOG / ObGyn


Dr Firman Abdullah SpOG/ OBGYN, Bukittinggi, Sumatera Barat ,Indonesia

Dr Firman Abdullah SpOG/ OBGYN,                              Bukittinggi, Sumatera Barat ,Indonesia

Bukittinggi , Sumatera Barat , Indonesia

Bukittinggi , Sumatera Barat  , Indonesia
Balaikota Bukittinggi

dr Firman Abdullah SpOG / OBGYN

dr Firman Abdullah SpOG / OBGYN

Ngarai Sianok ,Bukittinggi, Sumatera Barat.Indonesia

Ngarai Sianok ,Bukittinggi, Sumatera Barat.Indonesia

Brevet in Specialist Obstetric's & Gynecologist 1998

Brevet in Specialist Obstetric's & Gynecologist 1998
dr Firman Abdullah SpOG/ObGyn

Dokter Spesialis Kebidanan dan Penyakit Kandungan . ( Obstetric's and Gynaecologist ) . Jl.Bahder Johan no.227,Depan pasar pagi ,Tembok .Bukittinggi 26124 ,HP:0812 660 1614. West Sumatra,Indonesia

Sikuai Beach ,West Sumatra ,Indonesia

Sikuai Beach ,West Sumatra ,Indonesia

Fort de Kock, Bukittinggi