All the Small Things

It’s always interesting/entertaining/amusing when an animal does something we think is typically human; a dog riding a bike, a cat playing Candy Crush, a hamster surfing, and thanks to the internet we can see all these things, what a time to be alive. But the other week I was surprised to hear of an animal that did something no-one thought it could do and the repercussions of this could have a major impact on the world of endometriosis research.

This was a report I read documenting the first ever observed occurrence of a species of mouse (the spiny mouse to be precise) having a menstrual cycle. Up until now the only animals we were familiar with that menstruate were primates like chimps, gorillas and humans. Other animals have different reproductive cycles characterised by different hormonal and physical changes in their reproductive organs. Most other mammals, including mice, undergo an estrous cycle. The estrous cycle, like the menstrual cycle, is still regulated by hormones such as estrogen and progesterone, but the way these hormones vary throughout the respective cycles and the effect they have, differs.

The diagram below shows how the different hormones that regulate the function of the reproductive organs changes over the human menstrual cycle and the rat estrous cycle. If you want to see how these hormones work in humans I've covered it here.

Image source: http://pubs.niaaa.nih.gov/publications/arh26-4/images/emanuele3.gif

As you can see, there are quite obvious differences, but also some similarities between the two. The rat proestrus stage for example, is somewhat similar to the human day 14 and the rat day 4 resembles the human day 10, but overall they are quite distinct. In terms of the actual physical changes, menstrual animals shed their endometrium after each cycle if conception doesn’t occur. This, as some of you will all too painfully know, is having a period. Estrous animals on the other hand reabsorb the endometrium if conception doesn’t occur and, although some animals like dogs might bleed at certain times of their cycle, this is not an actual period, rather it is caused by estrogen making the blood vessels in the vagina ‘leaky’.

So the discovery of a menstrual cycle in the spiny mouse is surprising to say the least, but why is this relevant to endometriosis research? In order to study the way in which endometriosis works and develop new treatments we can grow endometriosis cells in the lab and experiment on them. Unfortunately this tells us nothing of the complexity of the disease or how endometriosis behaves in a living organism. For this researchers turn to animal models of the disease. This is usually achieved by ‘inducing’ endometriosis in a lab animal (typically estrous mice). However this is far from a perfect model of the disease. Inducing the disease in mice is achieved by transplanting either pieces of whole uterus, or pieces of the normal endometrium into the pelvis of the mice. Neither of these gives a true representation of human endometriosis, but it’s the closest thing researchers have at their disposal to study endometriosis (there are much more accurate primate models of endometriosis, but there are numerous reasons, such as cost, facilities and ethics, that prohibit the use of these animals in most research centres).

Another problem is that because normally mice don’t menstruate, the disease will not behave in mice the same way it behaves in humans due to different levels of hormones in the body. Mice can be made to have hormonal cycles like humans but this involves removing the ovaries and supplementing them with hormones and even this doesn’t represent the complexity of hormonal changes during the human cycle.

By using an animal that can menstruate, our models of endometriosis would become more accurate (in fact we may not need to induce endometriosis in these spiny mice if they develop it spontaneously, much more investigation is needed to observe this though). By extension the way we understand the disease in humans and the effectiveness with which we can develop new treatments will also become more accurate. Additionally, one of the goals that researchers work towards is reducing the number of animal experiments that are required to study any given disease. By using an animal that more closely resembles the human menstrual cycle, it would eliminate the need for performing additional procedures on lab mice to induce human-like hormonal cycles.

However, there is still a lot of work to find out exactly how similar the spiny mouse menstrual cycle is to the human one. The initial research that has been done has shown the spiny mouse cycle to be 9 days long. The human cycle is, on average, 28 days long and, interestingly, the pattern of progesterone levels in the blood across the spiny mouse cycle almost exactly matches that of the human cycle.

Although there is a lot of work still to be done investigating this unusual phenomenon, it not only has impact for endometriosis research, but also into other female reproductive conditions, like infertility, pregnancy and menstrual disorders. 

That Recurring Feeling

It’s that time of year again! Endometriosis awareness month is upon us and I’m a little bit late to the party with this first blog post, but I’ll be trying to get a few posts done this month to help raise awareness of the research most relevant to women with endo.

One of the major issues surrounding endometriosis therapy is surgical treatment for the disease. We all know that complete surgical removal of endometriosis offers the best hope of symptomatic relief (provided endometriosis is the only culprit contributing to symptoms, there are often other conditions associated with endo, like adenomyosis and interstitial cystitis, which can contribute to pelvic pain symptoms, that require differing treatment approaches). However, we don’t really have a lot of studies and empirical evidence in the area of surgical success for endometriosis surgery. Mostly it is down to self-reported numbers from surgeons themselves, which is fine, but it narrows the scope somewhat of how we can assess the success of surgery. The subject of this blog post will be looking at some recent publications concerning how endometriosis surgery is investigated and what we can learn about its effectiveness.

To begin with we have a study from China looking at the outcomes of laparoscopic surgery for endometriosis and how medical treatment after surgery affected the recovery. This study followed 199 with ovarian endometriosis for 3 years after laparoscopic surgery to remove all visible endometriosis and adhesions. These women were then divided into 3 groups depending on what treatment they had after surgery.

Group A were 43 women who only had surgical treatment

Group B were 47 women who had surgery and a GnRH drug afterward

Group C were 109 women who had surgery and mifepristone afterward.

So after the patients had been followed up after three years they were assessed to see whether their disease had returned (recurrence) or showed no signs of return (remission).

Group A – 58% were in remission, 28% had recurrence

Group B – 70% were in remission, 13% had recurrence

Group C – 61% were in remission, 25% had recurrence

You may notice that those percentages don’t really add up, that’s because there was another category of ‘improvement’ which was defined as symptoms remaining, but improving. However the numbers for this category weren’t reported in the paper. What this tells us is that there wasn’t a great deal of difference between women who had surgery alone and women who took mifepristone after surgery. There seemed to be a decrease in risk of recurrence with post-operative GnRH agonist therapy. This effect has also been seen in studies looking at surgery for endometriotic ovarian cysts. Unfortunately this therapy also comes with a long and unpleasant list of side-effects and is not suitable for long term therapy.

The next part was to look at the recurrence rate in women of different ages and different stages of endometriosis.

For the different age groups:

Women aged 25 or under, the recurrence rate was 0%

Women aged 25-29, the recurrence rate was 19%

Women aged 30-34, the recurrence rate was 25%

Women aged 34-38, the recurrence rate was 25%

When you look at these numbers you have to take into consideration  how many were in each age bracket. For example, there were only fourteen women in the 25 years old and under category, but there were one hundred and one in the 30-34 category. So although it looks as if there is more recurrence with age, it could be that there wasn’t a large enough number of patients in each group to give an accurate representation.

In terms of disease stage:

11% of women with stage II disease had recurrence

28% of women with stage III disease had recurrence

43% of women with stage IV disease had recurrence

So it seems as if there is a clear trend of increasing chance of recurring symptoms and disease at higher stages of endo. This could make sense as with advanced stages of endometriosis the pelvis can be congested with adhesions or obliteration of the cul-de-sac, making it difficult to see and excise all the endometriosis, leading to further operations.

The next is another study from China looking at deeply infiltrating endometriosis (DIE) and what are the outcomes for patients undergoing complete or incomplete removal of the disease. You may be wondering what the point of incomplete excision is. Surely you should always strive for complete removal of the disease? Because deeply infiltrating endometriosis, infiltrates deep (the clue’s in the name), this can mean that removing it all could cause damage the affected organs. This can be particularly dangerous where DIE affects the bowel and could lead to severe complications. So, incomplete removal of DIE is only performed where safety is an issue.

For this study there were 51 patients who underwent complete excision and 34 who underwent incomplete excision of their disease.  

Unlike the previous study this one included the location of the DIE as well. The most common sites of DIE in these patients were the uterosacral ligaments (one of the sets of ligaments that hold the uterus in place) and the posterior vaginal fornix (basically the ‘back of the vagina), which you can see labelled as ‘fornix’ in this diagram. Interestingly these two sites are pretty close to one another and as you move away from this area the incidence of DIE becomes less. For example, the number of cases of bladder DIE in this study was 4.3%, but the number of cases of uterosacral ligament DIE was 41.9%, so it would appear there is something about this region of the pelvis that is particularly prone to deeply infiltrating endometriosis. What exactly it is that causes the predisposition isn’t fully understood.  

After the women who underwent surgery were followed-up it was found that there was a substantial decrease in pain scores for women in both surgery groups. What will come as no surprise though is that the women who had complete excision had a much more dramatic decrease in pain scores compared to those who had incomplete excision. You will be similarly unsurprised to learn that the recurrence of disease was 3.9% in the complete excision group, compared to 35.3% in the incomplete excision group. In addition, the time it took for disease recurrence was almost 4 times longer in the complete excision group.

Leading on from this, the quality of life measurements showed that there were notable improvements in all aspects of quality of life (physical, psychological, social etc) in both surgery groups. The only significant difference though, was that women who had complete excision had a greater improvement in the ‘psychological’ component of the quality of life.  

Amongst all the women in this study who wished to conceive, no difference in the fertility outcomes between women who had incomplete or complete excision.

Like the previous study, this one also looked at how post-operative medical therapy can affect the outcome of surgery. This was, again, using GnRH therapy as the standard preoperative therapy, but only in the complete excision group who kept their ovaries. The authors divided patients into four groups depending on what and when they received their treatment:

Group 1) Did not receive GnRH before or after surgery (10 patients)

Group 2) Only received GnRH after surgery (30 patients)

Group 3) Only received GnRH before surgery (1 patient)

Group 4) Received GnRH both before and after surgery ((10 patients)

The authors then compared things like quality of life scores, pain scores and recurrence of disease between the groups to see if there was any effect the treatment regimens had on these outcomes. And the conclusion? There was no significant difference between any of these groups. The only difference they did observe was that those women who received GnRH therapy for 6 months (as opposed to 3 months) were less likely to need further surgery, but were more likely to develop osteoporosis. What is quite telling when you look at the numbers in the four groups is what preference doctors have for post-operative treatment. There are far more women who were given GnRH therapy after surgery who had not taken it before (group 2). It is fairly common practice to offer this type of therapy to women after surgery, yet this study seems to suggest (even though it is a fairly small study) that post-operative therapy makes very little, or no, difference to patient outcomes, but complete  excision of disease does benefit patients the most. Which I think is the take home message from this paper.          

Don't go Breaking my Heart

To my shame I haven’t managed to post much recently, normally I do a run of posts for Endometriosis Awareness Month in March but I haven’t been able to this year. To rectify this I’m going to be upping my posting frequency throughout the rest of the year as penance for my lack of posting thus far.

Chances are you may have seen a report from a recent piece of research about a link between endometriosis and an increase in risk in heart disease. It’s an attention grabbing article for sure, but what exactly is this new research telling us?

This week I’ll be going through the origin research article and dissecting exactly what the authors found.

This research was published in the journal of Circulation, Cardiovascular Quality and Outcomes in March of this year. The study enrolled 116,430 women in 1989 and followed them up over the course of 20 years with periodical questionnaires about their health. At the beginning of the study there were 5,296 women with laparoscopically confirmed endometriosis and 109,161 women without. By the end of the study the number of women with laparoscopically confirmed endometriosis had risen to 11,903.

At the beginning of the study when comparing things like age, BMI, contraceptive use, smoking, alcohol consumption, diet, and parents heart attack incidence, the groups of women with and without endo were very similar. Some real differences were noticeable when comparing the menopausal and surgical history of the two groups. More women in the endo groups were postmenopausal (14% vs 2%), had a hysterectomy (21% vs 4%), or had either one or both ovaries removed (17% vs 2%).

The main outcome of this study was to look for the rates of coronary heart disease (CHD) in these groups of women and compare them. In this case CHD was defined the occurrence of one or more of the following: heat attack (fatal or non-fatal), angina, coronary bypass graft surgery/angioplasty (repair or unblocking of a heart vessel)/stent (a device to keep the arteries to the heart open).

At the end of the study, in the endometriosis group, there were 69 cases of heart attack, 149 cases of angina, 91 cases of coronary bypass/angioplasty/stent and 207 cases of combined CHD.

After doing some fancy calculations and taking into account factors like age, smoking, diet etc the authors found that, overall, women with endometriosis were 63% more likely to have one of the CHD aspects than women without endo. When taking the individual CHD factors into account they found a 53% increase in heart attacks, 91% increase in angina and a 35% increase in coronary bypass/angioplasty/stent. Now those numbers seem pretty scary right? But they are relative. Consider the following example, if I tell you I’m going to increase your chance of getting struck by lightning by 100%, does that mean you are absolutely going to be struck by lightning? Nope, it would only mean that if I said I’m going to increase your chance of getting struck by lightning to 100%.

To work out how much your risk is increased you need to know the risk for the general population. Going back to our lightning example, the lifetime chance of getting hit by lightning (in the US) is 1 in 12,000 or 0.00008%. so if I increased your chance by 100% (which is the same as doubling it) your lifetime risk of being hit by lightning would still be only 0.00016%, still a really, really small chance. So it’s always worth knowing what the actual chance of having something happen to you is before worrying about an increase in risk. Unfortunately heart disease is not as rare as being hit by lightning, quite the opposite in fact.  Heart disease affects around 1 in 4 women and is the number #1 killer of men and women in most western countries. So whilst this study isn’t saying you are absolutely going to have a heart attack or angina etc, it does highlight that women with endometriosis appear to be at a higher risk of these things than the general population.

To tease out the effect this study divided the women into age groups of: under 40, 40-50, 50-55 and 55 and over. What they found was that women with endo in the 40 and under category were at a much higher risk of CHD.  Now this may be the case, but 40 and under is a quite a broad age range, particularly when you take into account that one of the age groups (50-55) was a very narrow range. There is a brilliant graph in the paper that shows how risk of CHD increases with age for women with and without endo. Unfortunately I can’t show it here without invoking the gods of copyright, which is a shame because this graph shows that at age 25, the risk of CHD for women with and without endo is pretty much the same. As age increases you can see risk of CHD does increase between the two groups. Just by looking at the graph itself the risk in CHD for endo sufferers appears most pronounced between the ages of 40 and 50. After age 55 the risk of CHD becomes the same again for both groups. So it is inaccurate to say that the risk of CHD is the same for a woman with endo who is 25 years old and a woman who is 40 years old.

The next question is, if this association exists, why? From the analysis that was done on this data, we know that factors like, diet, smoking, BMI (the common risk factors for CHD) were already taken into consideration, so there must be something else. One of the main contributing factors identified by this study is having a hysterectomy and oophorectomy. This effect has been observed in other studies too, which found that hysterectomy and oophorectomy before the age of 50 increases the risk of cardiovascular diseases. Women with endometriosis, particularly severe form of the disease, are more likely to have a hysterectomy and/or oophorectomy at a young age. It is thought that reduction of estrogen in the body brought on by oophorectomy can increase the risk of cardiovascular disease. In addition hysterectomy can sometimes lead to ovarian failure, which would have the same effect. This brings into question the safety and necessity of these procedures (oophorectomy in particular) as a method of treatment for endometriosis. It also brings into question the safety of some medical therapies for endo. Many of such therapies are based on the notion that reducing estrogen production in the body stops the endometriosis from growing. This can involve taking drugs that suppress ovarian production of estrogen, bringing on a menopause-like state. If indeed reduction in estrogen is linked to cardiovascular problems in women, then this form of medical treatment for endo could also be increasing the CHD risk this current study has observed. This paints a big neon sign pointing at the need for 1) more surgeons skilled at complete excision of endometriosis lesions and 2) the need for drugs that target endometriosis specifically and not have a whole body effect.

This study provides a good starting point for further work into endometriosis and CHD. In particular it would be interesting to see if increased CHD risk in women with endo is intrinsic (i.e. it is just something that comes along with endo) or modifiable (i.e. it is due to the aforementioned treatments). CHD is one of the biggest killers of women in the western world, so if the risk of CHD can be reduced in women with endo, then it is important for doctors, surgeons and drug companies to change their attitudes to how endometriosis should be treated.  

The Rhythm is Gonna Get You

Are you a morning person, no? Me neither. We all tend to have a rhythm to our days that we prefer; some of us prefer to go to bed at 9pm, some us at 2am. Your preference for patterns of waking and sleeping actually has a biological basis called the circadian clock. This is, essentially, your body’s internal clock (which is essentially the timed switching on and off of certain genes) and allows your body to anticipate events that happen repeatedly every day and produce the correct physiological response (like feeling sleepy or awake at certain times).   Our circadian clocks are controlled by a small part of the brain called the suprachiasmatic nucleus (SCN) located within the hypothalamus. The SCN is responsible for coordinating rhythms in the hypothalamus and the pituitary gland which in turn form part of the hypothalamatic-pituitary-gonadal (HPG) axis. This HPG axis is important for driving the rhythms of several organs in your body, but most importantly for the subject of this week’s blog post, it orchestrates the rhythms in the reproductive organs.

In women the regulation and rhythm of the menstrual cycle is a tightly controlled systems involving the HPG axis. Below is a diagram that summarises how the components all work together.

Interestingly several organs in the human body have their own distinct clock (called a peripheral clock) that are linked to the clock in the brain (the central clock). One such peripheral clock is in the ovaries. The ovaries, in concert with the brain, control the monthly variation in hormonal levels and the menstrual cycle. However, not only do levels of hormones change over the month, but also over the course of a day. A good example of this is that the monthly LH surge, mentioned above, requires a certain signal at the end of a resting period, which is why 80% of women have their LH surge around 8 a.m.  However, it is not fully understood (in humans at least) how the monthly and daily variations in hormone levels are regulated.

Altering the pattern of exposure to light, food and sleep can disrupt the body’s clock which can have negative effects ranging from feeling sluggish to serious illnesses (in the case of long term disruption). Disturbances in the normal cycle of waking/sleeping/eating etc have been suggested to cause alterations in hormone levels. Indeed some studies have shown that even partial sleep deprivation can lead to temporary increases in estrogen and luteinizing hormone, as well as altering the production of several other hormones. Another study showed that women with certain mutations in a gene that is turned on in an area of the brain that controls circadian rhythms leads to those women experiencing more miscarriages and less pregnancies than those women without the mutation. So clearly there is an important link between cycles in the brain and the reproductive system and disrupting one can adversely affect the other.

As modern society creates the necessity for longer and more varied working hours it is inevitable that some people will work at times that are out of synch with the ‘normal’ waking/sleeping rhythms we are used to. Disruption of the normal patterns of sleep because of shift working has been suggested to increase the risk of conditions such as cardiovascular disorders, gastrointestinal disorders, cancer and mental health conditions. So clearly there are negative health effects associated with shift work.

Of particular interest then are studies that have suggested that night shift work can increase the risk of having endometriosis, in some cases by as much as 50%. This same study found that there were no associations between endometriosis and mutations involving genes that regulate circadian rhythms, which leads me to believe the increase in risk was due to changes in hormones as a direct result of shift work. Now before I go any further there is a question I would like you to consider. Does night shift work actually increase the risk of developing endometriosis, or does night shift work increase the severity of endometriosis related symptoms making it more likely to be diagnosed? I very much doubt that night shift work can actually cause endometriosis, but it is certainly feasible that disruption of hormonal cycles by shift working could worsen the symptoms.

Indeed some studies have shown that, while levels of FSH and LH are not affected by night shift work, levels of estrogen are significantly increased, possibly due to a lengthening of the follicular phase of the menstrual cycle. Additionally melatonin, a hormone whose production is greatly affected by light/dark cycles but also has anti-estrogenic effects, has been shown to be reduced in shift workers. Perhaps as a result of this further studies demonstrated that shift work can cause alterations to menstrual cycle length and regularity.

Is there any way we can tie these effects to the symptoms of endometriosis? We know that when it comes to endometriosis, heightened levels of estrogen are bad. Pretty much all medical therapies for endo are based on reducing estrogen levels. Estrogen leads to increased growth and inflammatory action of endometriotic lesions.  Estrogen also increases the production of enzymes that produce chemical messengers called prostaglandins. Prostaglandins have several different functions, but in the uterus, they control the contractions of the uterine muscle, which are more commonly known as menstrual cramps. Excessive prostaglandin production can lead to severe and debilitating menstrual cramps (dysmenorrhoea) which is the most common symptom experienced by women with endometriosis.Therefore it could be that increased estrogen levels as a result of shift work could increase the severity of endometriosis associated dysmenorrhea.

I mentioned melatonin before as well and that was no mistake. There has been a lot of interest recently in melatonin as a treatment for endometriosis associated pain. Whilst most of the studies have only been lab based so far, some clinical trial data suggests that melatonin therapy could reduce the chronic pain associated with endometriosis as well as improve sleep quality.

In some ways the symptoms of endometriosis may be forming a vicious cycle. Some studies have shown that a substantial proportion of women with endo (of the cul-de-sac in the case of this study) experience sleep disturbances, insomnia and daytime fatigue. 

This goes a way to show how treating endometriosis should be viewed from many different angles to achieve maximum effectiveness. At present most medical therapies for endo are focussed on one particular aspect of the disease. A broader view of the disease and all of its strengths and weaknesses are needed before sufferers really see some benefit. 

Report from Juneau Biosciences

Way back in the distant and hazy past of 2009 I wrote about a company called Juneau Biosciences who were looking to recruit women with endometriosis for genetic research study. Well the results are in and they have released a preliminary report on what they have found.

The report states that they have found mutations in an imprinted gene called NLRP2 that are more common in women with endometriosis. What does this mean? Well, that will be the subject of todays blogpost.

First of all we need to understand what an imprinted gene is and why they are important. I’ve wrote before about genes, what they are and how they work, but what are imprinted genes? Normally a child inherits two ‘active’ copies of each gene, one from the mother and one from the father. However in some cases one of the genes, from either the father or the mother, can be switched off or ‘imprinted’ meaning that the child only has one working copy of the gene. This makes imprinted genes more ‘vulnerable’ because normally there would be two working copies of each gene, so if one copy gets mutated or stops working, there is a backup copy. With imprinted genes there is no backup, so if that gene has an important function (like preventing a cell becoming cancerous, for example) then there is a greater risk of a deleterious effect occurring.

Onto the next question, what does NLRP2 gene do and why is that relevant to endometriosis? The products of NLRP genes, in general, have a role in regulating the immune system and inflammation, which may be their most relevant known function in endometriosis. In particular NLRP2 has been shown to inhibit some key mediators of inflammation. Endometriosis is characterised by an increase in inflammation, both around the lesions and throughout the whole body (it is even thought that this might contribute to the fatigue many women with endometriosis experience). Also, I have written previously about how women with endo are at a higher risk of certain inflammatory and immune conditions, like inflammatory bowel disease, allergies and asthma to name a few. So finding alterations in a gene that is involved in inflammation and the immune system is potentially quite intriguing.

It could be tempting to suggest that this new finding could go some way to explaining why there is excess inflammation in endometriosis. However the sheer number of factors that contribute to the regulation of inflammation in endometriosis is huge (and still not fully understood) so exactly how significant a single factor is remains to be seen (in any case, the exact role of NLRP2 in general has been poorly characterised). Another point is that we need to know how these mutations in the NLRP2 gene affect the function of the protein it produces.

So there is a way still to go before this research can be translated into something relevant for the women living with endometriosis, but all progress needs a starting point, and this is a pretty good starting point.