LabEvoEndo Journal Club: Dana Ahern presents on PMDD

This is the third guest post of the LabEvoEndo Journal Club, a new series for the LabEvoEndo blog meant to highlight student contributions to the lab (first post here, second post here). The author is Honors Anthropology Junior Dana Ahern. Dana has been in my lab since her sophomore year.


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I’d like to start this blog post with a little background on progesterone. Progesterone is fascinating and it affects a lot more than just the menstrual cycle. We are starting to understand just how big of an impact progesterone has, and the research project I am working on is beginning to show some of the potential applications of progesterone in medicine. These applications include improving recovery time after traumatic brain injury and strokes, as well as helping us understand postpartum depression (PPD) and premenstrual dysphoric disorder (PMDD).

PMDD is similar in mechanism with postpartum depression, although the exact mechanism is still being researched. Some research suggests a hormonal influence, as allopregnanolone, an offshoot neuroregulator of progesterone, modulates GABA, which is a neurotransmitter related to mood and anxiety. Too much GABA results in increased anxiety and mood related issues, so it is a logical conclusion that the drop in progesterone during the late luteal phase of the menstrual cycle would result in excess GABA and therefore symptoms of PMDD.

The article we discussed looked at PET scans of 12 women with and 12 women without a diagnosis of PMDD, taking scans during the follicular phase and the late luteal phase of their menstrual cycles. The women in the study were screened for two months with mood surveys and then taken in for a day of PET scans and blood/urine samples, once during the follicular phase and once during the late luteal phase, when the symptoms of PMDD would have begun. PMDD can affect certain brain functions, so they were using the PET scan to detect brain dysfunction, such as one study the article looked at that showed frontal lobe dysfunction associated with PMDD.

The hormone measurements didn’t show anything significant and PET scans showed increased cerebellum activity from follicular to late luteal phase in PMDD women only. The cerebellum has many GABA receptors, which is a possible explanation of this, but in journal club, we wondered if it is the lack of modulation after a drop in the progesterone, or if there is something going on with the number of receptors, such as perhaps some sort of diminished sensitivity to allopregnanolone.

In journal club, we discussed some of the problems we had with this study. Someone brought up the small sample size and too few collection times for samples and scan. The fact that only one cycle was measured impacted the study. Kate also mentioned that blood hormones are a less useful measure than salivary hormone measurements. Finally, while the article states that the cause of PMDD is an overactive cerebellum in women suffering from PMDD, they never really reach a definite reason for what is causing the increased action. Ultimately, the article would have benefitted from examining the hormones more closely and more often.

Reference

Rapkin AJ, Berman SM, Mandelkern MA, Silverman DH, Morgan M, & London ED (2011). Neuroimaging evidence of cerebellar involvement in premenstrual dysphoric disorder. Biological psychiatry, 69 (4), 374-80 PMID: 21092938

LabEvoEndo Journal Club: Sophia Bodnar presents on cervical cancer

This is the second guest post of the LabEvoEndo Journal Club, a new series for the LabEvoEndo blog meant to highlight student contributions to the lab (first post here). The author is Anthropology Junior Sophia Bodnar. Sophia has been in my lab since her sophomore year.

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In light of recent acceptance to a summer program where I will be working in immunology, I wanted to choose an article relevant to reproductive health and immunology. The first place I turned to was the Journal of Reproductive Immunology. This seems like a copout, but I found a very interesting and recent article for the lab meeting entitled "Higher levels of cervicovaginal inflammatory and regulatory cytokines and chemokines in healthy young women with immature cervical epithelium" by Hwang et al in 2011.

It would be wonderful if human variation was always taken into account, particularly when discussing reproductive health. This is exactly what the researchers from UCSF aimed to do and why this article was a refreshing read. It is known that women aged 15-24 have the highest rates of Sexually Transmitted Infections (STIs) and is commonplace in the medical community to associate STIs with risky sexual behavior. The study looked at cervical epithelium, tissue that lines the cervix, and accompanying levels of cytokines and chemokines in order to show basic biological differences in immune function of the cervix between adults and adolescents to account for the predominance of STIs in younger women.

Thirty-two women aged 13-21 were selected. The women were placed in the immature group (>50% columnar epithelium) or mature cervical group (<5% columnar epithelium). In order to help you envision these epithelial cells, think of a single line of figure skaters (fragile, columnar cells) versus a stack of sumo wrestlers (strong, squamous cells). Younger women have more columnar cells and throughout development the columnar cells become squamous cells. This is why it is extremely important to take variation into account, because at any given point in a woman’s development the ratio of squamous to columnar cells varies.

Participants were excluded for pregnancy, surgery on the cervix, cervical dysplasia (grades above 1), taking immunosuppressive agents, or being symptomatic for genital complaints such as yeast. However, women were not excluded for having HPV because the researchers consider it a very common infection. The women filled out surveys detailing sexual and nonsexual behavior (number of partners, contraception, substance abuse, etc) and reproductive health history (previous pregnancies, menarche, etc). Colpophotagraphs were taken of the cervical epithelium and the researchers measured and rated maturity by counting pixels from the photograph. Women were evaluated only once, a strong drawback to this study as they are interested in the variation of the epithelium.

They found that ten of the cytokines and chemokine levels were higher in the immature group (one cytokine was excluded). Although other studies have previously looked at cervical levels of cytokines and chemokines, this study looked at levels of these cell-signaling protein molecules in relation to type of cervical epithelium. The authors note that having more columnar cells may be considered beneficial for young women as these cells are more easily damaged and likely to initiate an immune response, but this would mean that younger women are less prone toward infection. The researchers then go on to conclude that because these columnar cells are more prone to damage and thus immune response, chronic inflammation is more likely which could lead to greater rates of infection. This study only looked at the epithelium of healthy women, so it is important to examine women with various infections in the future. The epithelium and levels of cytokines/chemokines in women with STIs could lead to a better understanding or correlation between type of epithelium and its subsequent immune response and risk rate.

In our discussion of the article, it was mentioned that there are no “normal” levels of cytokine and chemokine levels with which to compare the researcher’s values. In the study this was referred to as a challenge. Perhaps there will never be established normative levels of these protein molecules, but so what? Our lab group concluded that this should not be considered a challenge and may be beneficial as we will simply have to take variation into account which is strongly lacking in the medical realm of reproductive health! We all agreed that this study should have been conducted over a longer period of time, and that the women should have had their epithelium photographed and correlating levels of cytokines/chemokines measured far more often. Because this study’s main focus was variation of the epithilium, it would make sense for these women to contribute visits over the course of their entire menstrual cycle whereas this study simply adjusted for days since last menstrual cycle. We also discussed that this study may be perceived as far more qualitative than quantitative. However, we acknowledged that the clarity of the cervical photos was great so quantitative measurements of the pixels could be taken seriously. It was also noted that these findings were not all that surprising. It seems commonsense that younger women have cells that are more easily damaged and that this could lead to chronic inflammation that poses negative risks. Although this may be true, I think that we should still applaud these authors for acknowledging variation throughout their research.

I also find it interesting that the most common cervical cancer is squamous cell carcinoma. Columnar cells may be damaged more easily, but it seems that squamous cells are more prone towards division and supporting abnormal growth. Most cases of cervical cancer are due in part to HPV infection, which in going along with the findings of this article, is more common in younger women with immature columnar cells. It would be interesting to see how the squamous cells in women with cervical cancer compare to the columnar cells of women with HPV. Perhaps cervical cancer, as a result of HPV, is more common in women who have more columnar cells than squamous cells, but the cancer arises in and prefers the squamous cells because they are less likely to initiate a strong immune response. In general, I appreciate how easy and enjoyable this article is to read. Hopefully more articles acknowledging variation, particularly when it comes to reproductive health, will be published as a result of this interesting finding.

Reference

Hwang LY, Scott ME, Ma Y, & Moscicki AB (2011). Higher levels of cervicovaginal inflammatory and regulatory cytokines and chemokines in healthy young women with immature cervical epithelium. Journal of reproductive immunology, 88 (1), 66-71 PMID: 21051089

LabEvoEndo Journal Club: Laura Klein presents on food allergies

This semester I'm trying out something a bit new -- I've encouraged my students to do quick write-ups of the papers they present in journal club in a way they think would be suitable for a blog post. This is to share what we're up to and give my brilliant students a chance to show their stuff.

This first guest post of the LabEvoEndo Journal Club is by Integrative Biology Honors Senior Laura Klein. Laura's been in my lab since her sophomore year.


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Welcome, readers! This blog post will start a series of posts by undergraduate lab members about the topics that LabEvoEndo currently finds interesting and are discussing in our weekly lab meetings. Usually, these will be related to our own current research projects and will somehow tie into the broader goals of the lab.

The other week, I spent a lot of time looking at some Polish survey data related to food allergies and food intolerances that Dr. Clancy and I collected last summer. This led me to thinking about the health implications of food allergies, so this week’s lab meeting article is "Food allergies in children affect nutrient intake and growth” by Christie et al from 2002. As the authors from this article are all medical professionals, I also wanted to supplement this article with some information about the daily social stressors that food allergies can have on individuals or families, so we also read excerpts about The Impacts of Food Allergies on the Quality of Life’ from Fernandez-Rivas and Miles “Food allergies: clinical and psychosocial perspectives.”

To give you some background for the study, 6-8% of American children will develop food allergies in the first 3 years of life, which also happens to be a critical period for growth. Of children with food allergies, most will have a single allergy to cow’s milk, eggs, peanuts, what, soy, tree nuts, or fish. Previous studies have found that children with cow’s milk allergies have a lower height/age ratio, though they did not propose a mechanism for this trend. One possibility is that elimination diets in general may reduce the amount of macro or micro nutrients available in the diet.

In this study, Christie et al. compared 98 children with and 99 children without food allergies (all about age 4) to determine if food allergies and elimination diets impact the growth and nutrient intake of children with food allergies. All children who were diagnosed with an allergy had been following a food elimination diet. The BMI, height-for-age, and weight-for-age for all children was compared to the CDC’s Health Statistics Growth Charts, and individuals were classified as having ‘potential undernutrition’ (<25th percentile), ‘adequate nutrition’ (25th-75th percentile), and ‘potential overnutrition’ (>75th percentile). Parents also completed three days of dietary intake records, which were compared with recommended dietary allowances.

The authors found that more children with allergies were categorized as having ‘potential undernutrition’ as determined by height-for-age than children without allergies. Additionally, a large number of these children had two or more allergies. Because the article only focused on height-for-age despite collecting many other measurements, we were skeptical that food allergies were the sole cause having low height-for-age. Some possibilities that we discussed but the study did not control for included premature birth and genetic influences on height or growth rates.

Another study result that generated much discussion was that more than 25% of the children in both the control and allergy groups were not getting the recommended daily intake levels of calcium and Vitamins D & E. We thought this was problematic, not necessarily for the health of the children, but for how recommended daily intake levels are defined. Children with milk allergies who consumed supplementary fortified beverages were more likely to be meeting all of the intake recommendations than even children without any dietary restrictions. Considering this, are the current guidelines a reflection of realistic intake for small children?

In the discussion, the authors introduce the possibility that ‘catch-up’ growth during puberty. They suggest that this could be many food allergies disappear as children mature, so foods can be reintroduced into the diet. However, we also suggested that early teenagers are usually simply hungrier than toddlers (as anyone who has been in a high school cafeteria knows). Greater consumption of calories may provide greater energy sources for growth. Also, by puberty, people usually have more access to food because they are able to prepare it themselves. Both of these situations could contribute to more available energy that could be used for growth.

Finally, we wrapped up our discussion talking about how different kinds of elimination diets have biological and social impacts. For members of our group with food allergies and intolerances, especially ones like nuts or gluten, group dinners can be a challenge because hosts can be unsure what to prepare and may only have one or two ‘safe’ dishes. This related to a problem brought up in both articles- that parents with children newly diagnosed with food allergies may unnecessarily restrict many foods because they are unsure what will cause an allergic reaction. This could cause an artificially limited availability or nutrients or calories. And, to tie this back to an earlier point, as children grow up and learn what foods to avoid, their teachers and friends’ parents may give them more freedom to choose a range of foods that are acceptable, instead of limiting them to one or two ‘safe’ foods.

A good analogy to this situation might be someone who has just decided to adopt a vegetarian diet. Younger people may at first be so focused on avoiding meat that they don’t pay attention to the nutrition content of their food. For example, cheese pizza and grilled cheese are meat-free, but don’t provide all protein, vitamins, and minerals needed for a complete diet. Also, young people who still live at home may have less control over the types of food bought or prepared. As one former vegetarian in the group told us, to balance all the portions of your diet takes a lot of effort and planning. She said she had never been more aware of what she was eating, but that it took time to figure out how to achieve a good balance of nutrients.

Stay tuned for updates and about brains and hormones!

References

Christie L, Hine RJ, Parker JG, & Burks W (2002). Food allergies in children affect nutrient intake and growth. Journal of the American Dietetic Association, 102 (11), 1648-51 PMID: 12449289

Fernandez-Rivas, M, & Miles, S (2007). Chapter 1. Food allergies: Clinical and Psychosocial Perspectives Plant Food Allergens DOI: 10.1002/9780470995174.ch1

Adolescent Menstrual Variation and Oral Contraceptives

The guest post below is by LEE alums Theresa and Ashley (currently skilled lab technicians planning their next steps in life!), with minimal editing by me. They did some fantastic research in their senior year and put together this blog post to teach the world about menstrual cycling in young girls. It's worth a read, and please comment with your thoughts!

Adolescent Menstrual Variation and Oral Contraceptives
By: Theresa Emmerling, Ashley Higgins and Kathryn Clancy

Many informational videos and documentaries have been produced to inform young women on the changes that they will experience during puberty and upon first menstruation, also called menarche. Unfortunately, these explanations are quite often generalizations that can lead to misrepresentations of what is normal. The information available to young women today stresses normative values for menstrual cycle length rather than the wide degree of variation characteristic of their cycles and may have an impact on adolescents’ future choices concerning oral contraceptives.

Figure 1 from Vihko and Apter 1984

Most women are taught that the menstrual cycle is twenty-eight days long, with ovulation occurring at day 14, and that only tiny deviations from this are healthy. Teens are taught to expect that every month after first menses they will experience menstruation for about 5 days; however, most adolescents do not experience the average menstrual cycle. Many adolescents experience longer menstrual cycles, with the luteal phase lasting anywhere between four to thirteen days (Apter, 1978), and anovulation, where an egg is not released from the ovary, is the primary characteristic of their cycles (Figure 1, Vihko and Apter 1984). The HPO axis is not an “on/off” switch that commences a string of twenty-eight day menstrual cycles at menarche. Rather, it takes time for the pituitary to respond to LH surges, which occurs only after multiple exposures to GnRH and estrogen (Apter et al, 1978; Apter, 1997). Therefore, it can take an adolescent up to five years to begin seeing something closer to the average twenty-eight day cycle (Figures 1 and 2, Vihko and Apter 1984).

Figure 2 from Vihko and Apter 1984

To be fair, the challenge is determining what the normal variation in an adolescent’s cycle is and what is due to pathology: environmental factors and pathologies may influence the menstrual cycle during development of the HPO axis. Numerous studies have tried to pathologize what we believe is normal variation in adolescent menstrual cycles, calling them “menstrual disturbances” or characterizing them as some level of amenorrhea (Morimatsu et al, 2009). As of yet, there is no conclusive evidence. Many studies find correlations between irregular menstrual cycles and pathology (Singer et al, 2009), but do not control for oral contraceptives (i.e., van Hoof et al, 2004), other environmental factors that may influence the HPO axis (Christo et al, 2008), or the study consists of adolescents with an already known pathology (El-Khayat, 2008). The American Academy of Pediatrics published a paper in 2006 stating that while the definition of amenorrhea is the absence of menses for six or more months, adolescents should be examined after only three months and "not reassured that it is 'normal' to have irregular periods in the first gynecological years" (p. 2247).

Given that it is more common, even normal, for adolescents to have very long menstrual cycles, we are concerned with what looks like stronger criteria for amenorrhea in exactly the population where it is expected (though the AAP paper discusses the low frequency of very long cycles, it also fails to mention the frequency of the pathology they worry is hidden in these long cycles). Further, more stringent criteria that misrepresents normal variation in this group of women serves only to frighten a population already concerned about normality and fitting in. For many, the teenage years are a time when standing out is to be avoided, and anything that makes one seem abnormal – an early or late age at menarche, long or short menstrual cycles – can be a stigma and cause stress. Educating young women on the reality, that this variation is what is normal, and that if anything the 28-day cycle is abnormal at this life stage, may prevent some of the considerable misunderstandings they have about their health. Understanding one's own body is an important step in making educated, healthy choices in the future. If most young women do not actually experience what they are taught is the average menstrual cycle, despite the fact that the reality is that wide variation in menstrual cycle length is the primary characteristic of post-menarcheal cycling, we are providing information that could lead them to feel abnormal and want to treat what is likely healthy variation.

A source of serious concern stems from what’s happening in the doctor’s office. Doctors, particularly in the United States, prescribe oral contraceptives at the slightest mention of deviation from the norm or evidence of a “menstrual disturbance.” In 79% of cases, health care providers will prescribe OCs at the patient’s request (Andrista et al, 2004). Less than 5% of doctors offer alternative forms of contraception to their patients in the US (Gerschultz, 2007), despite the fact that intrauterine devices, or IUDs, are more widely prescribed globally, and are both safe and effective. Future research questions should address whether doctors believe OCs are the best choice for women or that they the easiest method to prescribe; further, it is important to understand the decisions involved in prescribing these hormone treatments to adolescents. We have found evidence of doctors prescribing oral contraceptives to girls as young as twelve years of age (Cromer et al., 2008). Additionally, doctors prescribe OCs for adolescents who do not “live a lifestyle conducive to menstruation” (Sucato, 2002). We worry that ingesting cocktails of exogenous hormones have become a tool of convenience without regard to the possible trade-offs in their consumption. There is a large gap in the literature concerning adolescents and the long-term effects of OCs, yet doctors’ primary concern is that adolescents will accidentally skip a couple pills every pack (Omar et al, 2005).

Natural variation in adolescent hormone levels is a largely unexplored realm of science, with the same handful of decades-old articles cited repeatedly. Further research will yield not only a better understanding of this "time of change" but also less pathologizing of natural variation. However, until that research is published and generally accepted in the scientific and medical communities, the prescription of OCs for adolescents should weighted against the possible long-term impacts of priming an immature HPO axis with hormones.

References

American Academy of Pediatrics Committee on Adolescence, American College of Obstetricians and Gynecologists Committee on Adolescent Health Care, Diaz A, Laufer MR, & Breech LL (2006). Menstruation in girls and adolescents: using the menstrual cycle as a vital sign. Pediatrics, 118 (5), 2245-50 PMID: 17079600

Andrist LC, Arias RD, Nucatola D, Kaunitz AM, Musselman BL, Reiter S, Boulanger J, Dominguez L, & Emmert S (2004). Women's and providers' attitudes toward menstrual suppression with extended use of oral contraceptives. Contraception, 70 (5), 359-63 PMID: 15504373

Apter, D, Viinikka, L, Vihko, R. (1978). Hormonal pattern of adolescent menstrual cycles. J of Clin Endocrinol Metab 47: 944-954.

APTER, D. (1997). Development of the Hypothalamic-Pituitary-Ovarian Axis Annals of the New York Academy of Sciences, 816 (1 Adolescent Gy), 9-21 DOI: 10.1111/j.1749-6632.1997.tb52125.x

Christo, K., Cord, J., Mendes, N., Miller, K., Goldstein, M., Klibanski, A., & Misra, M. (2008). Acylated ghrelin and leptin in adolescent athletes with amenorrhea, eumenorrheic athletes and controls: a cross-sectional study Clinical Endocrinology, 69 (4), 628-633 DOI: 10.1111/j.1365-2265.2008.03237.x

Cromer BA, Bonny AE, Stager M, Lazebnik R, Rome E, Ziegler J, Camlin-Shingler K, & Secic M (2008). Bone mineral density in adolescent females using injectable or oral contraceptives: a 24-month prospective study. Fertility and sterility, 90 (6), 2060-7 PMID: 18222431

El-Khayat, H., Soliman, N., Tomoum, H., Omran, M., El-Wakad, A., & Shatla, R. (2008). Reproductive hormonal changes and catamenial pattern in adolescent females with epilepsy Epilepsia, 49 (9), 1619-1626 DOI: 10.1111/j.1528-1167.2008.01622.x

GERSCHULTZ, K., SUCATO, G., HENNON, T., MURRAY, P., & GOLD, M. (2007). Extended Cycling of Combined Hormonal Contraceptives in Adolescents: Physician Views and Prescribing Practices Journal of Adolescent Health, 40 (2), 151-157 DOI: 10.1016/j.jadohealth.2006.09.013

Morimatsu, Y., Matsubara, S., Watanabe, T., Hashimoto, Y., Matsui, T., Asada, K., & Suzuki, M. (2009). Future recovery of the normal menstrual cycle in adolescent patients with secondary amenorrhea Journal of Obstetrics and Gynaecology Research, 35 (3), 545-550 DOI: 10.1111/j.1447-0756.2009.01014.x

Omar H, Kives S, & Allen L (2005). Extended use of the oral contraceptive pill--is it an acceptable option for the adolescent? Journal of pediatric and adolescent gynecology, 18 (4), 285-8 PMID: 16171734

Singer, K., Rosenthal, A., & Kasa-Vubu, J. (2009). Elevated Testosterone and Hypergonadotropism in Active Adolescents of Normal Weight with Oligomenorrhea Journal of Pediatric and Adolescent Gynecology, 22 (5), 323-327 DOI: 10.1016/j.jpag.2008.12.010

Which is more safe: home birth or hospital birth?

You have probably seen the buzz about the recent American Journal of Obstetrics and Gynecology article (Wax et al 2010) on home birth safety, and the editorial in the Lancet that took the article’s shaky meta-analysis to crazytown: “Women have the right to choose how and where to give birth, but they do not have the right to put their baby at risk,” they write.

I have a lot of thoughts about this study and how it has been covered, by medical doctors and by the media. My main issues revolve around: 1) what these statistics mean from a personal versus a public health perspective, 2) maternal recovery and mortality, 3) the problem with criminalizing home birth, 4) the literature around birth experiences and the process of birth and resources for women, and 5) how we should look at this topic in the future.

What do these statistics mean?

According to the Wax et al (2010) meta-analysis, the difference in infant mortality between hospital births and home births is 0.2% versus 0.9% (other people have already done a nice take-down of the cherry-picking of older studies that have received significant criticism, yet were included). While headlines have screamed that this is a three-fold difference, it makes sense for us to pay attention to the absolute values. Infant mortality, in hospital or home births, is under one percent. We can’t even say that one in one hundred babies die in childbirth in developed countries any more (at least not as a whole – for now I’ll side-step some major differences relating to social disparities and race). If you are pregnant and considering where you want to give birth, I’m not sure how this slight difference could really sway you one way or the other. The problem is that the editors of the Lancet (and others) are conflating public health recommendations with personal recommendations… and shaming women in the process.

From a public health perspective, I suppose I can grudgingly understand why the difference in infant mortality in home versus hospital births matters. But you cannot take population-wide statistics and apply them to individuals. To do so is to ignore inter and intrapopulational variation, and to take a women’s decision about her body out of the context in which it should be understood.

Another thing to notice, Wax et al (2010) found that “neonatal outcomes of planned home births revealed less frequent prematurity, low birthweight, and assisted newborn ventilation.” So of the more than 99% of babies who were fine, home birth babies tended to be healthier. This of course could be a bias of who chooses a planned home birth versus someone who does not, so I am not assuming the directionality to be that the home birth predicts healthier kids. But I wouldn’t be surprised if further analysis showed both directions to be causal; that is, that women more likely to have healthier kids choose to plan a home birth, but also that because home births have fewer interventions those kids are more likely to be healthy.

What about maternal health?

What the Lancet editorial and Wax et al (2010) mention only briefly, is that for maternal mortality and morbidity in low-risk births, to me, home births (and, I would contend, birth center births) are the clear winner. By not being in the same room as epidural medicine (it’s right behind you in big cabinets, just waiting for you to say “ow”*), single beds with little room to maneuver, continuous fetal monitoring and an IV under your skin as soon as you’re admitted, you avoid interventions that often carry their own significant risks and precipitate a cascade of other interventions.

Many women in the US don’t want to give birth in hospitals because being in a hospital increases the risk of maternal mortality and morbidity and, perhaps more importantly, slows recovery time. I say the recovery time issue may be more important, because while the US is embarrassingly bad at keeping mothers alive, the numbers are still better than in infant mortality (though, obviously, this makes sense from a life history perspective). The US is ranked 40^th in the world in terms of maternal mortality – that means 39 countries do a better job keeping women alive during childbirth. Our incidence of maternal mortality is increasing, not decreasing, with the latest figures for 2008 being 17 deaths out of 100,000 births (Canada, for instance, has 7/100,000). Developed countries with higher rates of home birth have lower rates of maternal mortality.

Criminalizing home birth

Another problem I have is that part of the reason they cite home births as unsafe is that so few of them are staffed by certified midwives (only one third according to the Lancet editorial). The only reason more home births are not staffed by certified midwives is that organizations like the American Medical Association and others have lobbied to keep home births illegal in many states. Midwives cannot legally help a family give birth at home where I now live, in Illinois. Instead, I have been told by local homebirth supporters that there is an underground movement of lay midwives who try to help women stay out of hospitals, if it’s what they want. Is this a safe way to give birth if you are low-risk? Maybe, maybe not. I’m not sure I would be keen on a home birth that did not have a Certified Professional Midwife or Certified Nurse Midwife attending, who also had a good relationship to a doctor at the nearest hospital. But I also would not want to give low-risk birth in a hospital, even if I had a midwife, because of the major risks you incur just by stepping into a hospital (like infection). Women in states like mine are stuck between a rock and a hard place: give birth at home and risk not having someone with the right qualifications (and potentially face legal action), or risk giving birth under conditions where you may have interventions you don't want, and treated like something less than human (which I'll get to more in the next section).

By criminalizing home birth, medical doctors and their lobbyists force women who don’t want a medical birth to find less-than-perfect alternatives. So when Lancet editors and others criticize US women for not having the right people at their home births, I call shenanigans: they were complicit in making the laws that prohibit it in the first place.

Birth experiences in hospitals, birth centers and the home

There is a huge literature already on the medicalization and pathologization of femaleness, and I encourage you to devour it all, from Emily Martin’s The Woman in the Body to Robbie Davis-Floyd’s Birth as an American Rite of Passage.

So the other issue I want to make sure to include here is that pregnant patients don’t have the same rights as non-pregnant patients in a hospital (link to pdf), to refuse treatment, to leave, to contest a decision; hospitals can and do get court orders to force pregnant women to receive treatments they have refused. On the one hand, I can understand that sometimes decisions need to be made quickly during labor. On the other hand, I think there is a problem when we place a fetus’s rights above that of its mother’s. This recent story of police violence against a pregnant woman links to several problems with pregnant patient rights. Here are examples of court-ordered interventions. And here is more information on pregnant patient rights.

These are other reasons many women find the idea of a hospital birth frightening, and thus choose home birth or a birthing center. And if you read Davis-Floyd and others, you will see the interviews of women who have had hospital births how they were disempowered by the experience. This isn’t to say there aren’t many, many women who aren’t totally satisfied with hospital births, and would never consider home birth. It’s just to say that to acknowledge differences in infant mortality risk that are not necessarily meaningful to an individual making a decision about this, in the absence of all this other information, is disingenuous on the part of the editorial writers at the Lancet.

Future work on this topic

My daughter, just born at the birth center.

Rather than blaming the women who are trying to make the best decisions for themselves and their families and fetuses, medical doctors should be doing more to make different kinds of birth options available for women. In 2008, I gave birth in a birth center that was across the street from a hospital – the Cambridge Birth Center in Cambridge, MA. I had midwives, doulas, a labor and delivery nurse, my husband and sister to help me. I had a nice big king-size bed, a big tub to labor in, and just about every device you can think of to help me labor and push out the baby, from floating noodles for the tub, to a yoga ball, to a birthing stool. The time to get from the birth center to operating room if there was a complication was 4 minutes – the same amount of time had I been in the delivery room at the same hospital. This option is not available to me now, as a resident of the state of Illinois.

I’d like to see is re-analysis of the literature, where only home births with a certified midwife are analyzed. This is what is possible if we allow women more choices in where they give birth, so this is what we should examine. When we decriminalize women who don’t want the higher risk of epidurals or cesareans or infections, when we provide women more options for how and when to labor and give birth to their babies, and when we are honest with the failings of the medical system towards pregnant women and women in general, we’ll be heading in the right direction.

References

Wax, J., Lucas, F., Lamont, M., Pinette, M., Cartin, A., & Blackstone, J. (2010). Maternal and newborn outcomes in planned home birth vs planned hospital births: a metaanalysis American Journal of Obstetrics and Gynecology DOI: 10.1016/j.ajog.2010.05.028

Editorial staff (2010). Home birth--proceed with caution. Lancet, 376 (9738) PMID: 20674705


*What bothers me about epidurals and narcotics is not that women choose to use them during birth. I have given birth, I know how much it hurts and how it feels like the pain will never, ever end. I would never, ever begrudge a woman for choosing pain relief under those circumstances. What bothers me is that it is presented almost as a given in most hospital settings these days, since the medicine is already in the mother’s hospital room. There are many interventions one can give for pain that are not pharmaceutical, but few doctors are trained in these interventions. My midwives and doulas had so many tricks up their sleeves that even though I was in excruciating pain, I was able to remain in the birth center and have a totally normal, natural childbirth. I also want to be clear that if I hadn’t clearly stated I wanted this in my birth plan, they would not have worked so hard to make this possible. I wanted to be kept out of the hospital, kept away from the risk of infection and the risk of narcotics and the risk of cesareans and episiotomies, so they helped facilitate that within what was safe for me and my baby.

Premenstrual syndrome: understanding origin and variation

If you are female and post-menarcheal (that is, you’ve had your first period), you have probably had at least one person tell you that you are PMSing – either jokingly, or with an unpleasant edge. You may have expressed anger or irritability; you may have simply stood up for yourself. Or, you may have no idea what prompted the statement.

Maybe you were angered further by this comment, because you have never experienced any shifts in mood in your premenstrual phase (the several days leading up to your period). Maybe you were embarrassed because, whether or not the comment was at the time accurate, you do feel different right before your period. There is a lot of variation in the emotional experience of the premenstrual phase, from no changes, to dysphoria (that’s mild depression) or anger and irritability, increased creativity, well-being and happiness, to a whole host of other feelings. Part of this variation is a small minority of women who do experience negative symptoms strong enough to interfere with their daily lives, often called premenstrual syndrome. A syndrome refers to the association of symptoms and/or signs that often occur together, but that does not mean the etiology, or origin, of this syndrome is the same for everybody, and that does not mean everyone has the same disease if they have the same set of symptoms. This distinction is important because there are several syndromes out there relevant to women’s health (polycystic ovary syndrome and chronic fatigue syndrome come to mind), yet it is a classification that says, to me, “hey, we don’t know what causes this and in fact it may be a whole bunch of different things going on for different people, but this is what we have so far.”

Despite the fact that the experience of the premenstrual phase is varied, and the negative symptoms some number of women experience are categorized by a syndrome which does nothing to explain its etiology, there appear to be only two games in town in treating PMS: hormonal contraceptives (HCs) and selective serotonin reuptake inhibitors (SSRIs). And the most studied potential origin of PMS is progesterone withdrawal.

Now, a twenty-eight day, ovulatory menstrual cycle looks like this:

Notice the estradiol (that’s a kind of estrogen) peak just before ovulation, and the swell of progesterone (pro-gest, so supporting gestation should you conceive) in the second half of the cycle. Progesterone is secreted by the corpus luteum, which is left behind by the follicle that erupts from your ovary when you ovulate. If that egg is not fertilized, to wend its way through the fallopian tube and invasively implant its way through your endometrium, then that corpus luteum and progesterone doesn’t have much of a purpose. Without a signal that pregnancy has occurred, the corpus luteum degrades, progesterone concentrations decline, and your nicely primed endometrium sloughs off to be able to make another plush surface for the next attempt.

Allow me to briefly wax anthropological before moving on in my discussion of these premenstrual symptoms. This twenty eight day cycle with its nice estradiol peak and progesterone swell and ovulation smack dab in the middle? It’s pretty much a farce.












Those are real luteal progesterone concentrations from healthy, normal, reproductively-aged women from past studies of mine in one population of women in rural Poland (the follicular phase is the first half of your cycle, luteal phase the second). And the next image is from a whole bunch of other studies showing population variation in progesterone. Regular menstrual cycles, and yet luteal phases that range from 9-17 days. Mid-luteal progesterone concentrations varying by several orders of magnitude. You are looking at data ONLY of healthy women, in a homogenous rural environment, and ONLY cycles where they ovulated. And yet, look at all that variation.

So hormone concentrations, menstrual cycle lengths, experiences of the menstrual cycle and menses themselves, vary so much so that the idea that there is such a thing as one normal cycle for everyone is patently false. Women’s reproductive functioning only makes sense in the context of its environment, and if it is responding logically (high stress, low function, and vice versa), it’s pretty safe to say that woman is normal.

Okay, so back to PMS and progesterone withdrawal. You may have noticed something in the preceding images: progesterone concentrations decline in the days leading up to menses in all ovulatory cycles. In a physiological process with so much variation it’s hard to determine universals, this is one of them. So I suppose it makes sense, then, that researchers think progesterone withdrawal has something to do with premenstrual symptoms. Further, post-partum depression or the maternity blues occurs soon after parturition, when a mother goes from having huge quantities of progesterone (orders of magnitude higher than in a regular menstrual cycle) to zero, once that placenta that was making all that yummy progesterone is birthed.

At the same time, if these symptoms are not universal, if in fact some women have positive experiences of their premenstrual phase (and post-partum period), and to top it off progesterone withdrawal is a major feature of the menstrual cycle for most women… then perhaps the idea that progesterone withdrawal is the trigger only serves to further pathologize a population that is historically overpathologized and understudied. That is, does it make sense to consider a universal feature of the menstrual cycle the culprit for a set of symptoms that affect about five percent of the population?

Let’s unpack the data a bit more, then, and cut these folks some slack… at least to start. There are two kinds of work going on in the literature: the first is the identification of the mechanism of how progesterone withdrawal could be producing effects in the brain, and what most folks are focusing on is the fact that progesterone concentrations are tied to allopregnanalone (ALLO) concentrations in the brain (this makes sense, since ALLO is a kind of progestin and progesterone is its precursor). The second is to identify whether it is how rapidly progesterone declines that produces negative symptoms (this certainly does vary, if you look at how different women’s mid-luteal progesterone concentrations are), the overall concentration, or the estradiol to progesterone ratio.

Several studies indicate an association between decreased ALLO concentrations and premenstrual syndrome and post-partum depression [1-6]. Gracia et al [3] also note lower ALLO concentrations in those individuals suffering from PMS who are responsive to SSRI treatment, versus unresponsive individuals. So it does seem like those with lower concentrations of ALLO (and thus lower progesterone at that time as well) are perhaps more likely to have PMS. However, it also looks like reduced ALLO isn’t the only explanation for PMS: 1) only 63% saw improvement with SSRI treatment, and 2) the women in this study (n = 46) were grouped into tertiles by their ALLO concentrations, and the highest tertile (the women with the highest ALLO concentrations) did not show improvement. So it seems it is possible to have PMS without low ALLO relative to your population.

The second issue, that it may not be total levels of progesterone/ALLO triggering PMS symptoms but rather something about the rapidity of decline (an understandable hypothesis to start from given what we know of post-partum depression) is not as frequently studied. But I did find one study that examined this and managed to review a few articles I hadn’t yet found: Beckley and Finn [1] described work that suggested more rapid decline in progesterone concentrations was associated with depression-like behaviors in rodents, and themselves found similar results both when creating progesterone withdrawal conditions with supplementation, and when using finasteride (which inhibits progesterone metabolism; also known as Propecia).

I think the idea here and the results are tantalizing, but I am not yet confident in the mechanism. Part of the reason for this is that in human studies women were only sampled once per cycle in the luteal phase; often something like five plus or minus three days before menses. If you look at the graph above (this is the same as the second figure, just the mean and standard deviation instead of the concentrations of each individual), that six day spread (-8 to -2) shows a significant amount of time-dependent variation in progesterone concentrations. Further, progesterone is strongly influenced by age, energy balance, energy expenditure, and population (developmental conditions) [7-14], and I am unclear whether this variation has an impact on these results or can be controlled for in future work. One last point to consider is that social influences on perceptions of menstruation, as well as degree of social support, urban versus rural setting, and level of education, predict premenstrual syndrome and post-partum depression quite strongly [15-18]. We need to be careful when examining something that is a product of the interaction between biology and culture not to simply look at the concentration of a hormone to explain its etiology.







References
1. Beckley EH, & Finn DA (2007). Inhibition of progesterone metabolism mimics the effect of progesterone withdrawal on forced swim test immobility. Pharmacology, biochemistry, and behavior, 87 (4), 412-9 PMID: 17597197
2. Brinton RD, Thompson RF, Foy MR, Baudry M, Wang J, Finch CE, Morgan TE, Pike CJ, Mack WJ, Stanczyk FZ, & Nilsen J (2008). Progesterone receptors: form and function in brain. Frontiers in neuroendocrinology, 29 (2), 313-39 PMID: 18374402
3. Gracia CR, Freeman EW, Sammel MD, Lin H, Sheng L, & Frye C (2009). Allopregnanolone levels before and after selective serotonin reuptake inhibitor treatment of premenstrual symptoms. Journal of clinical psychopharmacology, 29 (4), 403-5 PMID: 19593190
4. Maguire, J., & Mody, I. (2008). GABAAR Plasticity during Pregnancy: Relevance to Postpartum Depression Neuron, 59 (2), 207-213 DOI: 10.1016/j.neuron.2008.06.019
5. Monteleone, P. (2000). Allopregnanolone concentrations and premenstrual syndrome European Journal of Endocrinology, 142 (3), 269-273 DOI: 10.1530/eje.0.1420269
6. Nappi, R. (2001). Serum allopregnanolone in women with postpartum “blues” Obstetrics & Gynecology, 97 (1), 77-80 DOI: 10.1016/S0029-7844(00)01112-1
7. Chapman, J., McIntyre, M., Lipson, S., & Ellison, P. (2009). Weight change and ovarian steroid profiles in young women Fertility and Sterility, 91 (3), 858-861 DOI: 10.1016/j.fertnstert.2007.12.081
8. ELLISON, P. (1993). Population variation in ovarian function The Lancet, 342 (8868), 433-434 DOI: 10.1016/0140-6736(93)92845-K
9. Ellison PT, Panter-Brick C, Lipson SF, & O'Rourke MT (1993). The ecological context of human ovarian function. Human reproduction (Oxford, England), 8 (12), 2248-58 PMID: 8150934
10. Jasieńska G, & Ellison PT (1998). Physical work causes suppression of ovarian function in women. Proceedings. Biological sciences / The Royal Society, 265 (1408), 1847-51 PMID: 9802241
11. Lager, C., & Ellison, P. (1990). Effect of moderate weight loss on ovarian function assessed by salivary progesterone measurements American Journal of Human Biology, 2 (3), 303-312 DOI: 10.1002/ajhb.1310020312
12. O'Rourke, M.T., S.F. Lipson, and P.T. Ellison, Ovarian function in the latter half of the reproductive lifespan. American Journal of Human Biology, 1996. 8(6): p. 751-759.
13. Panter-Brick C, & Ellison PT (1994). Seasonality of workloads and ovarian function in Nepali women. Annals of the New York Academy of Sciences, 709, 234-5 PMID: 8154716
14. Rosetta L, Harrison GA, & Read GF (1998). Ovarian impairments of female recreational distance runners during a season of training. Annals of human biology, 25 (4), 345-57 PMID: 9667360
15. Cénac A, Maikibi DK, & Develoux M (1987). Premenstrual syndrome in Sahelian Africa. A comparative study of 400 literate and illiterate women in Niger. Transactions of the Royal Society of Tropical Medicine and Hygiene, 81 (4), 544-7 PMID: 3445335
16. Chaturvedi SK, & Chandra PS (1991). Sociocultural aspects of menstrual attitudes and premenstrual experiences in India. Social science & medicine (1982), 32 (3), 349-51 PMID: 2024146
17. Marván ML, Díaz-Erosa M, & Montesinos A (1998). Premenstrual symptoms in Mexican women with different educational levels. The Journal of psychology, 132 (5), 517-26 PMID: 9729845
18. O'hara, M., & Swain, A. (1996). Rates and risk of postpartum depression—a meta-analysis International Review of Psychiatry, 8 (1), 37-54 DOI: 10.3109/09540269609037816

A new direction

One of the projects that is just getting off the ground in the Laboratory for Evolutionary Endocrinology is the study of hormonal contraception in young women. We will be exploring the science behind it -- when is it useful to young girls and women, when is it not, what future health implications does it have -- but also the relationships between health care providers and patients regarding this drug.

I am developing a hypothesis regarding when health care providers prescribe hormonal contraceptives. For now, and this is before I have delved into the literature on this, I would like to test the conditions under which it is prescribed for a health complaint, and the conditions under which the health complaint is further explored instead. My suspicion is that when a woman goes to her PCP with a reproductive health issue but is not currently interested in having children, she is often prescribed hormonal contraceptives. When a woman goes to her PCP with a reproductive health issue and is interested in having children, the possible health concern is explored.

I would like to test this hypothesis and others using an online survey in the coming months. As we work to put the survey together and obtain IRB approval, I would like to solicit your thinking. What kinds of questions should we ask? What population should we seek for answers? Should we survey doctors as well as patients? What hypotheses would you want to see tested?

Put your questions and comments in the comments section of this post. If I get a lot of clarification queries, I will post more information about what we're thinking to give you more ideas.

New Center for Gynepathology Research

Linda Griffith, PhD, Professor of Biological and Mechanical Engineering at MIT, opens a new Center for Gynepathology Research today. She has paired with Padma Lakshmi of Top Chef fame to cast light on endometriosis, a condition that is said to affect 10% of women (and probably many more). You can go to the Center site here, and read the Boston Globe article about the Center here.

Dr. Griffith is doing women all over the world a service with the opening of this new Center. She is pairing up with scholars and clinicians at MIT, Vanderbilt, Harvard, Cornell and Tufts. I'm very excited by the fact that people who do basic science will be working with clinicians on a disease whose research has not advanced that much in recent years. We have a few hypotheses about why women get endometriosis -- some genetic causes, inflammatory variables, and flowback of copious menstruation -- but so far little of this research has yielded results that help prevent endometriosis, or help treat it. This Center is an important step forward.

We anthropologists and evolutionary medicine folks happen to like evidence from the angle of global and ecological variation in a condition in order to set a baseline and understand pathology better. So here are some additional questions that I think we should be asking about endometriosis:

• What is the global range of variation in the presence of endometriosis in different populations? I'm not talking just about the debilitating symptoms, but the presence of endometrial tissue outside the uterus. And what is the range of variation for amount of endometrial tissue outside the uterus?
  
• What ecological/lifestyle factors are most commonly associated with endometriosis? I'm thinking not just energetic stress, but immunological stress, psychosocial stress, diet composition, etc.
  
• Are there less invasive ways to test for endometriosis (i.e., ultrasound) that will allow us to examine this disease in non-western or non-industrialized environments?
  
• How do daily ovarian hormone concentrations vary with endometriosis incidence?
  
• When and under what conditions does endometriosis co-occur with other gynepathologies, such as polycystic ovarian syndrome?

Evolution of Diseases of Modern Environments: my report now up

Many of you know that I attended a conference in Berlin last month entitled "Evolution and Diseases of Modern Environments" that was convened by Randolph Nesse and dove-tailed with the 350th World Health Summit. Ben Campbell (University of Wisconsin, Milwaukee) and I were the rapporteurs for our group "Early Development and Reproductive Function." Our report is now online at The Evolution and Medicine Review and can be found here.

A highlight to get you over there:

The fertility group’s main conclusion was that we need to bring our particular approach to variability to medicine.... The main problem we note is that we are uncomfortable making strong recommendations to medicine regarding reproductive health because we do not yet have enough baseline data of the normal range of variation within and between women and within and between populations. The focus in funding mechanisms is on basic molecular science and disease-focused science, and our discipline falls between these two extremes. Thus we first propose a greater emphasis on research that assesses normal variation in the following ways: longitudinal, repetitive sampling, an assessment of lifestyle factors, documentation of ethnic and geographic variation, and a focus on the major lifestyle transitions as these can be periods of major variability.... Finally, we wanted to point out that the population that the majority of our data is western and economically developed, and that they represent the most extreme and highest concentrations of ovarian hormones (and likely other indicators of reproductive function).

Go!

The latter half of the cycle and the endometrium

Because it's been a while since any of us has blogged, I thought I would post a quick teaser regarding our AAPA and HBA abstracts. Following up from a recent paper in Anthropological Science on rural Polish women, we have found in Canadian women that luteal phase length is correlated to endometrial thickness through the implantation window. The implantation window is six to twelve days after ovulation (the middle of the cycle), and the average luteal phase is twelve to sixteen days long (the second half of the menstrual cycle). Before menses endometrial thickness begins to decline, so as you might expect, those with shorter luteal phases have thinner endometria through the window of implantation.

For now this simply demonstrates variation in endometrial thickness through what has been demonstrated to be the period during which the vast majority of implantations occur. This also means that there is significant variation in luteal phase length, even in ovulatory cycles in well-fed, urban women, in a way that has the potential to be biologically meaningful. Are there factors that are producing variation in luteal phase length (timing of menstruation) that in turn impact endometrial thickness through the implantation window? Or is the endometrium breaking down early for some reason (not a timing so much as a maintenance issue), leading to the shorter luteal phase? How much of this variation is genetic?

This demonstrates that normal women are incredibly variable in their reproductive physiology. Thus, deviation from the "norm" is not immediately suggestive of pathology.

What does this anthropologist think about hormonal birth control? Part V

Parts I, II, III, IV and V.

This is the final act of the five part series on hormonal contraception. In the first acts, I shared with you some of my opinions regarding the way the pill and assorted hormonal contraceptives are marketed, described some current literature on pill use, normal variation in reproductive function, and possible behavioral changes associated with reproductive hormones in contracepting and noncontraceptiving women. Today, I’ll continue to be a bit opinionated and offer up some alternatives to hormonal contraception. As always, keep in mind that I am not a medical doctor, but a biological anthropologist. This is not medical advice, and while it is research blogging this is not original research or even a review article. This is a series of blog posts written by an academic with enough expertise and enough of a strong opinion that she feels like sharing it.

So what should I use for birth control?

I would like to first mention that there are some very good reasons to use hormonal contraceptives. If you have difficulties using barrier methods and are in a monogamous sexual relationship where you are not concerned about STDs, if you are interested both in birth control and are seeking to reduce PMDD symptoms or moderate acne, or if you wish to use birth control without your partner knowing about it, hormonal contraceptives are a good choice. If you do not have any risk factors for the side effects associated with them, if you have a BMI under 25, if you were born and raised in an industrialized nation, then hormonal contraceptives are fine too if you want to use them.

However (and this is a big however), it is worth considering what parts of hormonal contraceptives have not been adequately studied (in terms of behavior, cognition, libido), and what you are comfortable supporting in terms of pharmaceutical companies. Further, water purification treatments have a lot of trouble getting hormones out of water because they are so small – this means they are getting into our rivers (I wish I had the source for this, but a colleague borrowed my textbook on this over a year ago and I haven’t gotten it back – the only article I can find on my desktop right now is Huang and Sedlack 2001 which is worth reading, but doesn’t address everything). This is not me wagging my finger – we are all complicit in any number of things every day of the week. I indulge in Cherry Coke Zero from the vending machine down the hall from my office every now and again, and I know that Coca Cola is not the best company. I own clothes from companies that use sweatshops. I am not always a shining example of righteousness. So please do not misunderstand this point as my saying that those who use hormonal contraceptives are not good people.

When none of the choices are especially appealing, and reproductive decisions are largely in the hands of women, where women are in fewer positions of power (except as the not-as-influential-as-you-may-think consumer), it is irrational to judge any individual woman for her reproductive decisions.

So, what kind of contraceptive should YOU use? I do hope you know about all of the available barrier methods: condoms, female condoms, diaphragms, sponges, and spermicides. For someone in a monogamous, long term relationship I think diaphragms are a great choice and with a little practice very easy to use. Another method worth considering is the IUD, or intrauterine device. This is one of the most popular birth control methods across the globe, but because there were some health scares with their use in the very early stages (think several decades ago) people in the US don’t use them. IUDs these days are very safe, and if you find a doctor who doesn’t want to put one in you because you haven’t had a kid yet, find another doctor (this was the advice given to me by a medical doctor, by the way). The no-IUDs-until-kids thing is a very old, conservative stance that doesn’t match the evidence for the usefulness of IUDs.

The reason I wanted to write this series of posts on hormonal contraception was not to universally advise against it. Rather, I was interested in promoting some of the research around it (and perhaps demonstrating some of the holes in what we know), in ways the average user of contraception may not think to look into. Population variation, cultural conceptions of menstruation, BMI, behavior, libido and cognition, as well as what companies profit from our use of these products, are all worth considering, alongside the convenience of taking a pill to avoid pregnancy. In time I hope we’ll be able to say more about behavioral or cognitive effects of hormonal contraception, especially given the huge numbers of women out there who use it.

References

Huang C, & Sedlack DL (2001). Analysis of estrogenic hormones in municipal wastewater effluent and surface water using ELISA and GC/MS/MS Environmental Toxicology and Chemistry, 20 (1), 133-139