Posts Tagged ‘testosterone’

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Research about marijuana and fertility is limited but some previous studies suggested that it might harm semen quality. Smoking of any type is also known to be a risk factor for male infertility. So, men who have smoked cannabis are expected to have worse measures of fertility but the data from a recent study suggested the opposite. The finding contradicts all conventional knowledge on how weed affects sperm. This may be because previous research typically focused on men with drug abuse history but this present study simply asked men if they had smoked more than two joints in their life.


Analysis of 1,143 semen samples from 662 men collected between 2000 and 2017 at the Fertility Clinic at Massachusetts General Hospital showed that those who had smoked weed at some point in their life had a mean sperm concentration of 62.7 million sperm per milliliter (mL) of ejaculate, while men who avoided marijuana entirely had mean concentrations of 45.4 million/mL. Added to this only 5% of weed smokers had sperm concentrations below the 15 million/mL threshold the World Health Organization has set for a “normal” sperm count, versus 12% of men who never smoked marijuana.


The study has some imperfections such as the participants are not necessarily representative of the general population. They were predominantly college educated men with a mean age of 36, and were all seeking treatment at a fertility center. Further research is needed to support the findings. Two possibilities are put forward by the researchers as the reason behind such data. The first is that low levels of marijuana could have a positive effect on the endocannabinoid system, the neurotransmitters in the nervous system that bind to cannabinoid receptors, and are known to regulate fertility. The second is that may be weed-smokers are just bigger risk takers and men with higher testosterone levels and thus have better sperm count.


But, there’s certainly no medical recommendation to smoke weed as a fertility treatment but this study, at least, suggests that a little marijuana doesn’t hurt and might benefit sperm production in some way. But, the researchers specified that their finding does not necessarily mean that smoking cannabis increases the chances of fatherhood.




Read Full Post »

Testosterone treatment improved primarily sexual function than walking or vitality in older men with low testosterone levels

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

A preliminary study of testosterone therapy in older men with low levels of the hormone and clinical conditions to which low testosterone might contribute, found that restoring levels to those of healthy young men improved sexual function. Treatment had a smaller effect on other aspects of health, such as the ability to walk or the sense of vitality.


A high proportion of older men have testosterone levels well below those found in healthy younger men. In most cases, these low levels are not due to diseases known to affect testosterone levels, such as testicular or pituitary conditions. Many of these men also have symptomatic problems that could be related to low testosterone, including diminished sexual function, decreased mobility and fatigue.


For a long time, there has been interest in whether testosterone is an appropriate therapy for aging-related conditions in men. This study clarifies questions about some of its potential benefits. The study did not find a pattern of increased cardiovascular disease risk. Clarifying the risks requires further study.


Participants included 790 men age 65 and older with serum testosterone levels consistently well below the average for young healthy men. They were randomized to receive testosterone gel applied to the skin or a placebo gel daily. Serum testosterone concentration was measured at one, two, three, six, nine and 12 months. The men were also closely monitored for prostate and cardiovascular problems. In addition to low testosterone, the presence of at least one of three conditions (low sexual function, difficulty in walking or low vitality) was required for eligibility to participate in the T Trials (Testosterone Trials).


  • Sexual function — In men with low sexual function, testosterone treatment increased sexual activity, sexual desire and erectile function more than placebo treatment.


  • Physical function — In men with difficulty in walking, testosterone treatment did not significantly affect walking ability, as measured by the distance they could walk in six minutes (a common test of walking ability). However, in all men, walking speed and distance did improve among those who received testosterone compared with placebo.


  • Vitality — In the group of men with symptoms of low vitality and fatigue, testosterone treatment did not significantly affect fatigue symptoms, but had modest favorable effects on mood.


The trials’ results indicate that, for older men with low sexual function, testosterone treatment can contribute to improved function. In contrast, though, the results don’t indicate that testosterone treatment for older men with low walking ability or vitality will improve these conditions to a great extent. Older men should consult their physicians if considering a testosterone treatment.




Read Full Post »

Hormone Therapy [9.6]

Writer and Curator: Larry H. Bernstein, MD, FCAP

The structure of this article is as follows:

9.6.1 Hormone Treatment Fights Prostate Cancer

9.6.2 Diabetes and Cardiovascular Disease During Androgen Deprivation Therapy for Prostate Cancer

9.6.3 Breast Cancer and Hormone Therapy

9.6.4 Hormone Therapy and Different Ovarian Cancers

9.6.5 Chemotherapy versus hormonal treatment in platinum- and paclitaxel-refractory ovarian cancer: a randomised trial of the German Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) Study Group Ovarian Cancer


9.6.1 Hormone Treatment Fights Prostate Cancer

By R. Morgan Griffin

Hormone therapy for prostate cancer has come a long way in the past few decades. Not so long ago, the only hormonal treatment for this disease was drastic: an orchiectomy, the surgical removal of the testicles.

Now we have a number of medications — available as pills, injections, and implants — that can give men the benefits of decreasing male hormone levels without irreversible surgery.

“I think hormonal therapy has done wonders for men with prostate cancer,” Stuart Holden, MD, Medical Director of the Prostate Cancer Foundation.

Hormone therapy for prostate cancer does have limitations. Right now, it’s usually used only in men whose cancer has recurred or spread elsewhere in the body.

But even in cases where removing or killing the cancer isn’t possible, hormone therapy can help slow down cancer growth. Though it isn’t a cure, hormone therapy for prostate cancer can help men with prostate cancer feel better and add years to their lives.

On average, hormone therapy can stop the advance of cancer for two to three years. However, it varies from case to case. Some men do well on hormone therapy for much longer.

The idea that hormones have an effect on prostate cancer is not new. The scientist Charles Huggins first established this over 60 years ago in work that led to his winning the Nobel Prize. Huggins found that removing one of the main sources of male hormones from the body — the testicles — could slow the growth of the disease.

“This procedure worked dramatically,” says Holden, who is also director of the Prostate Cancer Center at Cedar Sinai Medical Center in Los Angeles. “Before, these men were confined to bed and wracked with pain. Almost immediately afterwards, they improved.”

Huggins found that some types of prostate cancer cells androgens — to grow. Testosterone is one kind of androgen. About 90% to 95% of all androgens are made in the testicles, while the rest are made in the adrenal glands.

Hormone therapy for prostate cancer works by either preventing the body from making these androgens or by blocking their effects. Either way, the hormone levels drop, and the cancer’s growth slows.

In 85% to 90% of cases of advanced prostate cancer, hormone therapy can shrink the tumor.

However, hormone therapy for prostate cancer doesn’t work forever. The problem is that not all cancer cells need hormones to grow. Over time, these cells that aren’t reliant on hormones will spread. If this happens, hormone therapy won’t help anymore, and your doctor will need to shift to a different treatment approach.

There are two basic kinds of hormone therapy for prostate cancer. One class of drugs stops the body from making certain hormones. The other allows the body to make these hormones, but prevents them from attaching to the cancer cells. Some doctors start treatment with both drugs in an effort to achieve a total androgen block. This approach goes by several names: combined androgen blockade, complete androgen blockade, or total androgen blockade.

Here’s a rundown of the techniques.

  • Luteinizing hormone-releasing hormone agonists (LHRH agonists.)These are chemicals that stop the production of testosterone in the testicles. Essentially, they provide the benefits of an orchiectomy for men with advanced prostate cancer without surgery. This approach is sometimes called “chemical castration.” However, the effects are fully reversible if you stop taking the medication.Most LHRH agonists are injected every one to four months. Some examples are Lupron, Trelstar, Vantas, and Zoladex. A new drug, Viadur, is an implant placed in the arm just once a year.

    Side effects can be significant. They include: loss of sex drivehot flashes, development of breasts (gynecomastia) or painful breasts, loss of muscle, weight gain, fatigue, and decrease in levels of “good”cholesterol.

    Plenaxis is a drug that’s similar to LHRH agonists. However, because it can cause serious allergic reactions, it’s not used that often.

  • Anti-androgens. LHRH agonists and orchiectomies only affect the androgens that are made in the testicles. Thus they have no effect on the 5% to 10% of a man’s “male” hormones that are made in the adrenal glands. Anti-androgens are designed to affect the hormones made in the adrenal glands. They don’t stop the hormones from being made, but they stop them from having an effect on the cancer cells.The advantage of anti-androgens is that they have fewer side effects than LHRH agonists. Many men prefer them because they are less likely to diminish libido. Side effects include tenderness of the breasts, diarrhea, and nausea. These drugs are also taken as pills each day, which may be more convenient than injections. Examples are CasodexEulexin, and Nilandron.

    In some cases, starting treatment with an LHRH agonist can cause a “tumor flare,” a temporary acceleration of the cancer’s growth due to an initial increase in testosterone before the levels drop. This may cause the prostate gland to enlarge, obstructing the bladder and making it difficult to urinate. It’s believed that starting with an anti-androgen drug and then switching to an LHRH agonist can help avoid this problem. In patients with bone metastases, this “flare” can lead to significant complications such as bone pain, fractures, and nerve compression.

    Strangely, if treatment with an anti-androgen doesn’t work, stopping it may actually improve symptoms for a short time. This phenomenon is called “androgen withdrawal,” and experts aren’t sure why it happens.

  • Combined Androgen Blockade. This approach combines anti-androgens with LHRH agonists or an orchiectomy. By using both approaches, you can cut off or block the effects of hormones made by both the adrenal glands and the testicles. However, using both treatments can also increase the side effects. An orchiectomy or an LHRH agonist on its own can cause significant side effects like a loss of libido, impotence, and hot flashes. Adding an anti-androgen can cause diarrhea, and less often, nauseafatigue, and liver problems.
  • Estrogens. Some synthetic versions of female hormones are used for prostate cancer. In fact, they were one of the early treatments used for the disease. However, because of their serious cardiovascular side effects, they’re not used as often anymore. J. Brantley Thrasher, MD, a spokesman for the American Urological Association and chairman of urology at the University of Kansas Medical Center, says they’re usually used only after initial hormone treatments have failed. Examples of estrogens are DES (diethylstilbestrol), Premarin, and Estradiol.
  • Other Drugs. Proscar (finasteride) is another drug that indirectly blocks an androgen that helps prostate cancer cells grow. Depending

on the case, doctors sometimes use other anticancer drugs like Nizoral (ketoconazole) and Cytadren (aminoglutethimide.)

  • Orchiectomy. The surgical removal of the testicles was the earliest form of hormone therapy for prostate cancer. However, the procedure is permanent. As with LHRH agonists, side effects can be significant. They include: Loss of sex drive, hot flashes, development of breasts (gynecomastia) or painful breasts, loss of muscle, weight gain, fatigue, and decrease in levels of “good” cholesterol.

Hormone therapy for prostate cancer can cause osteoporosis, which can lead to broken bones. However, treatment with bisphosphonates — like ArediaFosamax, and Zometa — may help prevent this condition from developing.

Hormone (androgen deprivation) therapy for prostate cancer

Hormone therapy is also called androgen deprivation therapy (ADT) or androgen suppression therapy. The goal is to reduce levels of male hormones, called androgens, in the body, or to stop them from affecting prostate cancer cells.

The main androgens are testosterone and dihydrotestosterone (DHT). Most of the body’s androgens come from the testicles, but the adrenal glands also make a small amount. Androgens stimulate prostate cancer cells to grow. Lowering androgen levels or stopping them from getting into prostate cancer cells often makes prostate cancers shrink or grow more slowly for a time. But hormone therapy alone does not cure prostate cancer.

Hormone therapy may be used:

  • If the cancer has spread too far to be cured by surgery or radiation, or if you can’t have these treatments for some other reason
  • If your cancer remains or comes back after treatment with surgery or radiation therapy
  • Along with radiation therapy as initial treatment if you are at higher risk of the cancer coming back after treatment (based on a high Gleason score, high PSA level, and/or growth of the cancer outside the prostate)
  • Before radiation to try to shrink the cancer to make treatment more effective

Several types of hormone therapy can be used to treat prostate cancer. Some lower the levels of testosterone or other androgens (male hormones). Others block the action of those hormones.

Luteinizing hormone-releasing hormone (LHRH) analogs

These drugs lower the amount of testosterone made by the testicles. Treatment with these drugs is sometimes calledchemical castration or medical castration because they lower androgen levels just as well as orchiectomy.

Even though LHRH analogs (also called LHRH agonists or GnRH agonists) cost more than orchiectomy and require more frequent doctor visits, most men choose this method. These drugs allow the testicles to remain in place, but the testicles will shrink over time, and they may even become too small to feel.

LHRH analogs are injected or placed as small implants under the skin. Depending on the drug used, they are given anywhere from once a month up to once a year. The LHRH analogs available in the United States include leuprolide (Lupron®, Eligard®), goserelin (Zoladex®), triptorelin (Trelstar®), and histrelin (Vantas®).

When LHRH analogs are first given, testosterone levels go up briefly before falling to very low levels. This effect is called flare and results from the complex way in which LHRH analogs work. Men whose cancer has spread to the bones may have bone pain. If the cancer has spread to the spine, even a short-term increase in tumor growth as a result of the flare could compress the spinal cord and cause pain or paralysis. Flare can be avoided by giving drugs called anti-androgens for a few weeks when starting treatment with LHRH analogs. (Anti-androgens are discussed further on.)

Degarelix (Firmagon®)

Degarelix is an LHRH antagonist. LHRH antagonists work like LHRH agonists, but they reduce testosterone levels more quickly and do not cause tumor flare like the LHRH agonists do.

This drug is used to treat advanced prostate cancer. It is given as a monthly injection under the skin. The most common side effects are problems at the injection site (pain, redness, and swelling) and increased levels of liver enzymes on lab tests. Other side effects are discussed in detail below.

Abiraterone (Zytiga®)

Drugs such as LHRH agonists can stop the testicles from making androgens, but other cells in the body, including prostate cancer cells themselves, can still make small amounts, which can fuel cancer growth. Abiraterone blocks an enzyme called CYP17, which helps stop these cells from making androgens.

Abiraterone can be used in men with advanced castrate-resistant prostate cancer (cancer that is still growing despite low testosterone levels from an LHRH agonist, LHRH antagonist, or orchiectomy). Abiraterone has been shown to shrink or slow the growth of some of these tumors and help some of these men live longer.

This drug is taken as pills every day. This drug doesn’t stop the testicles from making testosterone, so men who haven’t had an orchiectomy need to continue treatment with an LHRH agonist or antagonist. Because abiraterone also lowers the level of some other hormones in the body, prednisone (a cortisone-like drug) needs to be taken during treatment as well to avoid certain side effects.

Drugs that stop androgens from working


Androgens have to bind to a protein in the cell called an androgen receptor to work. Anti-androgens are drugs that bind to these receptors so the androgens can’t.

Drugs of this type, such as flutamide (Eulexin®), bicalutamide (Casodex®), and nilutamide (Nilandron®), are pills taken daily.

Anti-androgens are not often used by themselves in the United States. An anti-androgen may be added to treatment if orchiectomy, an LHRH analog, or LHRH antagonist is no longer working by itself. An anti-androgen is also sometimes given for a few weeks when an LHRH analog is first started to prevent a tumor flare.

Anti-androgen treatment can be combined with orchiectomy or an LHRH analog as first-line hormone therapy. This is called combined androgen blockade (CAB).

9.6.2 Diabetes and Cardiovascular Disease During Androgen Deprivation Therapy for Prostate Cancer

Nancy L. KeatingA. James O’Malley and Matthew R. Smith
JCO Sep 20, 2006; 24(27):4448-4456

Purpose Androgen deprivation therapy with a gonadotropin-releasing hormone (GnRH) agonist is associated with increased fat mass and insulin resistance in men with prostate cancer, but the risk of obesity-related disease during treatment has not been well studied. We assessed whether androgen deprivation therapy is associated with an increased incidence of diabetes and cardiovascular disease. Patients and Methods Observational study of a population-based cohort of 73,196 fee-for-service Medicare enrollees age 66 years or older who were diagnosed with locoregional prostate cancer during 1992 to 1999 and observed through 2001. We used Cox proportional hazards models to assess whether treatment with GnRH agonists or orchiectomy was associated with diabetes, coronary heart disease, myocardial infarction, and sudden cardiac death. Results More than one third of men received a GnRH agonist during follow-up. GnRH agonist use was associated with increased risk of incident diabetes (adjusted hazard ratio [HR], 1.44; P < .001), coronary heart disease (adjusted HR, 1.16; P < .001), myocardial infarction (adjusted HR, 1.11; P = .03), and sudden cardiac death (adjusted HR, 1.16; P = .004). Men treated with orchiectomy were more likely to develop diabetes (adjusted HR, 1.34; P < .001) but not coronary heart disease, myocardial infarction, or sudden cardiac death (all P > .20). Conclusion GnRH agonist treatment for men with locoregional prostate cancer may be associated with an increased risk of incident diabetes and cardiovascular disease.

9.6.3 Breast Cancer and Hormone Therapy

There are certain hormones that can attach to breast cancer cells and affect their ability to multiply. The purpose of hormone therapy, also called endocrine therapy, is to add, block, or remove hormones.

With breast cancer, the female hormones estrogen andprogesterone can promote the growth of some breast cancer cells. Therefore in some patients, hormone therapy is given to block the body’s naturally occurring estrogen to slow or stop the cancer‘s growth.

There are two types of hormone therapy for breast cancer.

  • Drugs that inhibit estrogen and progesterone from promotingbreast cancer cell growth.
  • Drugs or surgery to turn off the production of hormones from the ovaries.

Faslodex, a estrogen receptor antagonist, binds to estrogen receptors and blocks their effects on cancer cells. Given as an injection, the drug is for HER2-positive metastatic disease in postmenopausal women who have already tried anti-estrogen therapy. Common side effects of Faslodex include:

  • Injection site pain
  • Nausea and vomiting
  • Loss of appetite
  • Weakness, fatigue
  • Hot flashes
  • Cough
  • Muscle, joint, and bone pain
  • Constipation
  • Shortness of breath

Zoladex and Lupron for Breast Cancer

Zoladex and Lupron are drugs that stop the production of estrogen by the ovaries. They are used in premenopausal women for the treatment of estrogen sensitive breast cancer.

Side effects of Zoladex and Lupron include:

  • Fluid retention
  • Hot flashes
  • Irregular menstrual periods
  • Pain at the injection site

Hormone-sensitive breast cancer cells contain proteins known as hormone receptors that become activated when hormones bind to them. The activated receptors cause changes in the expression of specific genes, which can lead to the stimulation of cell growth.

To determine whether breast cancer cells contain hormone receptors, doctors test samples of tumor tissue that have been removed by surgery. If the tumor cells contain estrogen receptors, the cancer is called estrogen receptor-positive (ER-positive), estrogen-sensitive, or estrogen-responsive. Similarly, if the tumor cells contain progesterone receptors, the cancer is called progesterone receptor-positive (PR- or PgR-positive). Approximately 70 percent of breast cancers are ER-positive. Most ER-positive breast cancers are also PR-positive (1).

Breast cancers that lack estrogen receptors are called estrogen receptor-negative (ER-negative). These tumors are estrogen-insensitive, meaning that they do not use estrogen to grow. Breast tumors that lack progesterone receptors are called progesterone receptor-negative (PR- or PgR-negative).

Hormone therapy (also called hormonal therapy, hormone treatment, or endocrine therapy) slows or stops the growth of hormone-sensitive tumors by blocking the body’s ability to produce hormones or by interfering with hormone action. Tumors that are hormone-insensitive do not respond to hormone therapy.

Hormone therapy for breast cancer is not the same as menopausal hormone therapy or female hormone replacement therapy, in which hormones are given to reduce the symptoms of menopause.

Several strategies have been developed to treat hormone-sensitive breast cancer, including the following:

Blocking ovarian function: Because the ovaries are the main source of estrogen in premenopausal women, estrogen levels in these women can be reduced by eliminating or suppressing ovarian function. Blocking ovarian function is called ovarian ablation.

Ovarian ablation can be done surgically in an operation to remove the ovaries (called oophorectomy) or by treatment with radiation. This type of ovarian ablation is usually permanent.

Alternatively, ovarian function can be suppressed temporarily by treatment with drugs called gonadotropin-releasing hormone (GnRH) agonists, which are also known as luteinizing hormone-releasing hormone (LH-RH) agonists. These medicines interfere with signals from the pituitary gland that stimulate the ovaries to produce estrogen.

Examples of ovarian suppression drugs that have been approved by the U.S. Food and Drug Administration (FDA) are goserelin (Zoladex®) and leuprolide (Lupron®).

Blocking estrogen production: Drugs called aromatase inhibitors can be used to block the activity of an enzyme called aromatase, which the body uses to make estrogen in the ovaries and in other tissues. Aromatase inhibitors are used primarily in postmenopausal women because the ovaries in premenopausal women produce too much aromatase for the inhibitors to block effectively. However, these drugs can be used in premenopausal women if they are given together with a drug that suppresses ovarian function.

Examples of aromatase inhibitors approved by the FDA are anastrozole (Arimidex®) and letrozole (Femara®), both of which temporarily inactivate aromatase, and exemestane (Aromasin®), which permanently inactivates the enzyme.

Blocking estrogen’s effects: Several types of drugs interfere with estrogen’s ability to stimulate the growth of breast cancer cells:

  • Selective estrogen receptor modulators (SERMs) bind to estrogen receptors, preventing estrogen from binding. Examples of SERMs approved by the FDA are tamoxifen (Nolvadex®), raloxifene (Evista®), andtoremifene (Fareston®). Tamoxifen has been used for more than 30 years to treat hormone receptor-positive breast cancer.Because SERMs bind to estrogen receptors, they can potentially not only block estrogen activity (i.e., serve as estrogen antagonists) but also mimic estrogen effects (i.e., serve as estrogen agonists). Most SERMs behave as estrogen antagonists in some tissues and as estrogen agonists in other tissues. For example, tamoxifen blocks the effects of estrogen in breast tissue but acts like estrogen in the uterus and bone.
  • Other antiestrogen drugs, such as fulvestrant (Faslodex®), work in a somewhat different way to block estrogen’s effects. Like SERMs, fulvestrant attaches to the estrogen receptor and functions as an estrogen antagonist. However, unlike SERMs, fulvestrant has no estrogen agonist effects. It is a pure antiestrogen. In addition, when fulvestrant binds to the estrogen receptor, the receptor is targeted for destruction.

There are three main ways that hormone therapy is used to treat hormone-sensitive breast cancer:

Adjuvant therapy for early-stage breast cancer: Research has shown that women treated for early-stage ER-positive breast cancer benefit from receiving at least 5 years of adjuvant hormone therapy (2). Adjuvant therapy is treatment given after the main treatment (surgery, in the case of early-stage breast cancer) to increase the likelihood of a cure.

Adjuvant therapy may include radiation therapy and some combination of chemotherapy, hormone therapy, and targeted therapyTamoxifen has been approved by the FDA for adjuvant hormone treatment of premenopausal and postmenopausal women (and men) with ER-positive early-stage breast cancer, andanastrozole and letrozole have been approved for this use in postmenopausal women.

A third aromatase inhibitorexemestane, is approved for adjuvant treatment of early-stage breast cancer in postmenopausal women who have received tamoxifen previously.

Until recently, most women who received adjuvant hormone therapy to reduce the chance of a breast cancer recurrence took tamoxifen every day for 5 years. However, with the advent of newer hormone therapies, some of which have been compared with tamoxifen in clinical trials, additional approaches to hormone therapy have become common (35). For example, some women may take an aromatase inhibitor every day for 5 years, instead of tamoxifen. Other women may receive additional treatment with an aromatase inhibitor after 5 years of tamoxifen. Finally, some women may switch to an aromatase inhibitor after 2 or 3 years of tamoxifen, for a total of 5 or more years of hormone therapy.

Decisions about the type and duration of adjuvant hormone therapy must be made on an individual basis. This complicated decision-making process is best carried out by talking with an oncologist, a doctor who specializes in cancer treatment.

Treatment of metastatic breast cancer: Several types of hormone therapy are approved to treat hormone-sensitive breast cancer that is metastatic (has spread to other parts of the body).

Studies have shown that tamoxifen is effective in treating women and men with metastatic breast cancer (6).Toremifene is also approved for this use. The antiestrogen fulvestrant can be used in postmenopausal women with metastatic ER-positive breast cancer after treatment with other antiestrogens (7).

The aromatase inhibitors anastrozole and letrozole can be given to postmenopausal women as initial therapy for metastatic hormone-sensitive breast cancer (89). These two drugs, as well as the aromatase inhibitor exemestane, can also be used to treat postmenopausal women with advanced breast cancer whose disease has worsened after treatment with tamoxifen (10).

Neoadjuvant treatment of breast cancer: The use of hormone therapy to treat breast cancer before surgery (neoadjuvant therapy) has been studied in clinical trials (11). The goal of neoadjuvant therapy is to reduce the size of a breast tumor to allow breast-conserving surgery. Data from randomized controlled trials have shown that neoadjuvant hormone therapies—in particular, aromatase inhibitors—can be effective in reducing the size of breast tumors in postmenopausal women. The results in premenopausal women are less clear because only a few small trials involving relatively few premenopausal women have been conducted thus far.

No hormone therapy has yet been approved by the FDA for the neoadjuvant treatment of breast cancer.

9.6.4 Hormone Therapy and Different Ovarian Cancers

Lina Steinrud Mørch, Ellen Løkkegaard, Anne Helms Andreasen, Susanne Krüger Kjær, Øjvind Lidegaard
Am J Epidemiol. 2012; 175(12):1234-1242

Postmenopausal hormone therapy use increases the risk of ovarian cancer. In the present study, the authors examined the risks of different histologic types of ovarian cancer associated with hormone therapy. Using Danish national registers, the authors identified 909,946 women who were followed from 1995–2005. The women were 50–79 years of age and had no prior hormone-sensitive cancers or bilateral oophorectomy. Hormone therapy prescription data were obtained from the National Register of Medicinal Product Statistics. The National Cancer and Pathology Register provided data on ovarian cancers, including information about tumor histology. The authors performed Poisson regression analyses that included hormone exposures and confounders as time-dependent covariates. In an average of 8.0 years of follow up, 2,681 cases of epithelial ovarian cancer were detected. Compared with never users, women taking unopposed oral estrogen therapy had increased risks of both serous tumors (incidence rate ratio (IRR) = 1.7, 95% confidence interval: 1.4, 2.2) and endometrioid tumors (IRR = 1.5, 95% confidence interval: 1.0, 2.4) but decreased risk of mucinous tumors (IRR = 0.3, 95% confidence interval: 0.1, 0.8). Similar increased risks of serous and endometrioid tumors were found with estrogen/progestin therapy, whereas no association was found with mucinous tumors. Consistent with results from recent cohort studies, the authors found that ovarian cancer risk varied according to tumor histology. The types of ovarian tumors should be given attention in future studies.


Ovarian cancer is the most lethal of gynecologic cancers. Unfortunately, little is known about its etiology. In recent meta-analyses, investigators have concluded that women taking postmenopausal hormone therapy (HT) have an increased risk of ovarian cancer compared with never users.[1, 2] Two large prospective studies, the Million Women Study and Danish Sex Hormone Register Study, found an overall increased risk of 30%–40%.[3, 4]

Less is known about the association between hormone use and the risk of different histologic subtypes of epithelial ovarian cancer. Other risk factors for ovarian cancer have been found to differ between mucinous and nonmucinous ovarian tumors, supporting the hypothesis of different etiologies.[5, 6]However, previous studies on HT and different types of ovarian tumors were mainly case-control studies, and the numbers of cases were small, especially for mucinous tumors.[1, 7–10] Most prospective cohort studies either did not examine tumor type[1, 4] or had incomplete information on histology.[11]

Recently, Danforth et al.[12] found that estrogen-only therapy (ET) was more strongly associated with the risk of endometrioid tumors than with the risk of other types of epithelial tumors in the Nurses’ Health Study (NHS). The Million Women Study found that with HT use, the highest risk was for serous tumors, whereas there was a lower risk of mucinous tumors.[3] Knowledge about the associations between HTs and subtypes of ovarian cancer will add to the understanding of how HT acts as a promoter of ovarian cancer carcinogenesis. Moreover, if different types of ovarian tumors are to be viewed as separate diseases, that fact should be considered when creating the study designs for future research. Therefore, the aim of the present study was to explore the risks of HT associated with different histologic types of ovarian cancer.

The study cohort was linked to the National Register of Medicinal Product Statistics using participants’ personal identification numbers as the key identifiers. The National Register of Medicinal Product Statistics includes information on the date of the redeemed prescriptions and the specific Anatomical Therapeutic Chemical code, dose, number of packages, defined daily doses, and route of administration (tablet, patch, gel, etc.) The specific Anatomical Therapeutic Chemical codes included in the present study have been described previously.[13]

The information on initiation of HT use (i.e., redeemed prescriptions) was updated daily for each individual during follow-up. The prescribed defined daily doses were used to determine the length of use. We included 4 months after the expiration of the prescription in all records of hormone exposure to account for any delay in recorded diagnoses in Danish registers, prolonged HT use for those taking less than the defined daily dose prescribed, and minor latency time. Thus, gaps between prescriptions of less than 4 month were filled prospectively; that is, a woman was classified as user of the drug at a given point in time if the dispensed supply from the last redemption had not run out or if it had run out within the last m days (where m is the allowed gap length).[14]

Because HT is likely to act as a promoter of ovarian cancer carcinogenesis with a yet unknown latency time, women currently taking hormones were categorized by the regimen that they took for the longest period during the study period. These variables were time varying; that is, if a woman began a new HT regimen, she would be recategorized if and when the time taking that regimen exceeded the amount of time she took the prior categorization HT regimen. The length of use was calculated as the time spent taking all systemic treatments during the study period. Whether a woman had taken hormones before 50 years of age but within the 11-year study period was accounted for in the hormone status categories, and the amount of time for which she took the hormones was accounted for in the duration of use category. The HT categories were HT use (never, past, current nonvaginal HT use, or other current use (i.e., current use of vaginal ET or a hormone intrauterine device)); hormone formulation (ET, estrogen/progestin therapy (EPT), or other (i.e., tibolone, raloxifene, progestin only, or vaginal estrogen)); hormone regimen (cyclic EPT, continuous EPT, or other); route of administration (oral ET, oral EPT or tibolone, dermal ET, dermal EPT, or other); duration of HT in years (never, current, 0.01–4 years, 4.01–7 years, or >7 years or use of vaginal ET or a hormone intrauterine device); and time since last use among former users (never, current, 0.01–2 years, 2.01–4 years, 4.01–6 years, or >6 years or use of vaginal ET or a hormone intrauterine device).

Ovarian Cancer Cases

Until December 31, 2002, we used the Danish Cancer Register to identify cases of primary invasive ovarian cancers and their histologies, using the International Classification of Diseases for Oncologytopography code 183.0 and morphology codes ending with a 3. At time of the present study, information from January 2003 had not been updated in the Danish Cancer Register. Thus, from 2003 onward, the Pathology Register was used for case findings and information on histology. The invasive epithelial tumors were classified as serous (codes M84413, M84603, M84613, and M90143), endometrioid (codes M83803 and M83813), mucinous (codes M84703, M84803, and M90153), clear-cell (codes M83103 and M83133), adenocarcinoma not otherwise specified (code M81403), or epithelial not otherwise specified (codes M80203, M80703, M81303, M85603, M89333, M89803, and M90003). Nonepithelial invasive tumors and borderline tumors were not included. Eight women for whom we did not have histologic information were excluded. Information on the stages of disease was available from the Danish Cancer Register until December 31, 2002.

From 1995 to 2005, a total of 909,946 perimenopausal and postmenopausal women with no previous cancer or removal of ovaries accumulated 7.3 million person-years of observation, corresponding to an average follow-up period of 8.0 years. The number of incident malignant epithelial ovarian cancers during the study period was 2,681. Of these, 1,336 were serous tumors, 377 were endometrioid tumors, 293 were mucinous tumors, 159 were clear-cell tumors, 115 were nonspecified epithelial tumors, and 401 were adenocarcinomas not otherwise specified. At the end of follow up, 63% of the women remained never users of HT, 22% were previous users, and 9% were current users. Compared with never users, hormone users were more likely to have undergone a hysterectomy (18.0% versus 6.2%) or unilateral salpingo-oophorectomy (5.7% versus 1.9%), to have been sterilized (8.4% versus 5.4%), and to be parous (80.8% versus 75.2%). The characteristics of the study population have been published previously.[4]

Compared with never users, current users of hormones had an increased risk of serous tumors (incidence rate ratio (IRR) = 1.7, 95% confidence interval (CI): 1.5, 1.9) and of endometrioid tumors (IRR = 1.7, 95% CI: 1.3, 2.2). Current use of hormones was not associated with the risk of mucinous or clear-cell tumors (Figure 1). The incidence rate ratios for serous ovarian cancer increased with duration of hormone use (0.01–4 years, IRR = 1.5, 95% CI: 1.3, 1.8; 4.01–7 years, IRR = 1.7, 95% CI: 1.4, 2.1; and >7 years, IRR = 2.1, 95% CI: 1.6, 2.8). The incidence rate ratios for other types of epithelial ovarian cancer were not consistently associated with duration of use (Figure 2).

(Enlarge Image)

Figure 1.

Incidence rate ratios of epithelial ovarian cancers associated with current use of hormone therapy, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

(Enlarge Image)

Figure 2.

Incidence rate ratios of epithelial ovarian cancers associated with durations of hormone therapy in years, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Risk estimates for clear-cell cancer are not shown because there were few cases. Bars, 95% confidence interval.

Time Since Hormone Use

We found increased incidence rate ratios for serous ovarian cancers for a period of up to 2 years after cessation of HT. Thereafter, the risk approached that observed in never users. For endometrioid tumors, the risk was not significantly increased after cessation of HT (Figure 3).

(Enlarge Image)

Figure 3.

Incidence rate ratios of serous and endometrioid ovarian cancers associated with time since last hormone therapy use in years, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

Estrogen Therapy

Compared with never users, women on unopposed ET had an increased risk of serous tumors (IRR = 1.7, 95% CI: 1.4, 2.1) and a tendency toward an increased risk of endometrioid tumors (IRR = 1.4, 95% CI: 0.9, 2.1). In contrast, the risk of mucinous tumors was decreased (IRR = 0.3, 95% CI: 0.1, 0.8). No association was found between ET and the risk of clear-cell tumors (IRR = 0.6, 95% CI: 0.2, 1.5) (Figure 4).

(Enlarge Image)

Figure 4.

Incidence rate ratios of epithelial ovarian cancers associated with hormone therapy, Danish Sex Hormone Register Study, 1995–2005. A) Estrogen-only therapy; B) estrogen/progestin therapy. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

Women on oral ET had a statistically significantly increased risk of endometrioid tumors (IRR = 1.5, 95% CI: 1.0, 2.4), and the risks for serous, mucinous, and clear-cell tumors were similar to the risks found for all ET. Because the risk associations between transdermal ET and ovarian cancers were based on a few cases, the data are not shown. Vaginal estrogen alone was associated with an increased risk of serous tumors (IRR = 1.4, 95% CI: 1.1, 1.9), whereas no associations were found with endometrioid, mucinous, or clear-cell tumors (data not shown).

Combined Therapy

Women on combined EPT had increased incidence rate ratios for serous tumors (IRR = 1.6, 95% CI: 1.4, 1.9) and endometrioid tumors (IRR = 2.0, 95% CI: 1.5, 2.6), whereas no associations were found with mucinous or clear-cell tumors (Figure 4). Similar risk associations were found among women on oral EPT. Because there were few cases, data for transdermal EPT are not shown.

Duration of HT

The incidence rate ratios for serous ovarian cancer increased with increased duration of ET and after 7 years reached an incidence rate ratio of 2.9 (95% CI: 1.9, 4.3). The risks for endometrioid ovarian cancer were similar for all durations of ET (Table 1).

Among women on cyclic EPT, the risk of endometrioid ovarian cancer was increased by 70%–140%, whereas the risk was not increased among women on continuous EPT. The risks for serous ovarian cancer were similar regardless of the duration of cyclic or continuous EPT (Table 1). Results from crude and adjusted analyses were almost identical (data not shown).

Stage of Disease

Overall, the associations between HT and risks of different ovarian tumors did not change after adjustment for the stage of disease (Table 2). Although the analyses were slightly weakened by a lower number of cases, the results roughly showed similar incidence rate ratios across the stages of disease (Table 2).


The present large cohort study suggests that there is a differential influence of HT on different subtypes of ovarian cancer. Hormone users had an excess risk of serous and endometrioid tumors but not of mucinous and clear-cell cancers of the ovaries. Both combined EPT and unopposed ET were associated with increased risks of serous ovarian cancer. Furthermore, cyclic EPT and oral ET were associated with increased risks of endometrioid ovarian cancer. In contrast, no HT was associated with risk of clear-cell ovarian cancer, and women who had used ET had a decreased risk of mucinous ovarian cancer.

Serous Ovarian Cancer

Two large prospective cohort studies, the NHS and the Million Women Study, also found an increased risk of serous ovarian cancer among hormone users.[3, 12] In accordance with our finding, the Million Women Study reported an approximately 50% increased risk with HT.[3] The NHS supports our finding that increasing duration of ET is associated with increasing rate ratios for serous ovarian cancer.[12]

Endometrioid Ovarian Cancer

Although the Million Women Study found no association between any HT and the risk of endometrioid ovarian cancer, we found a 70% increased risk.[3] The NHS found a 50% increased risk of endometrioid tumors after 5 years of ET.[12] In our study, women on oral ET had an up to 2-fold increased risk of endometrioid tumors. Because ET increases the risk of endometrial cancer[15] and endometrioid ovarian tumors are histologically similar to endometrial tissue (16), it seems likely that ET acts through similar biologic mechanisms in the development of endometrioid ovarian cancer, a hypothesis suggested by Danforth et al..[12]

Furthermore, the present study suggests that women on cyclic EPT have an increased risk of endometrioid ovarian cancer, whereas the risk is not increased in women on continuous EPT. Only one study addressed the risk of endometrioid ovarian tumors among women on cyclic versus continuous EPT, and those investigators were not able to demonstrate an increased risk with cyclic or continuous EPT.[7] With regard to the development of endometrial cancer, the increased risk has been found to be confined to women on cyclic EPT.[15] Thus, it is possible that cyclic EPT acts through similar biologic mechanisms in the development of endometrioid ovarian cancer.

Mucinous Ovarian Cancer

Compared with women who were never prescribed HT, women on ET had a 70% decreased risk of mucinous ovarian cancer. The Million Women Study also found a decreased risk of approximately 30% with the use of HT.[3] A few other studies have also suggested that HT is associated with a decreased risk of mucinous ovarian cancer.[12, 17, 18] One group of mucinous tumors is similar to endocervical epithelium and another is similar to colonic epithelium.[16] Both HT in general and ET specifically have been found to decrease the risk of colon cancer.[19, 20] It therefore seems plausible that ET could also decrease the risk of mucinous ovarian cancer. Risch et al.[5] were the first to suggest different etiologies for mucinous and nonmucinous ovarian cancers, and a recent Danish study supported this hypothesis by suggesting significant differences in the risk between mucinous and nonmucinous tumors.[6]


Using the same data as in current study, Mørch et al.[4] found a 40% increase in the overall risk of ovarian cancer in current users of hormones, regardless of the duration and type of HT. However, in the present study, the risk of serous ovarian tumors increased with increasing durations of hormone use. This association was more pronounced among women using ET. After 7 years, the risk of serous ovarian cancer had increased 3-fold among women using ET compared with never users. On the other hand, restricting the analysis to mucinous tumors showed a decreased risk among women using ET. Thus, important information about a differential impact of HT, HT types, and associations with duration of hormone use are not described when different ovarian tumors are examined as a combined outcome.

Moreover, the clarification of the different associations between HT and subtypes of ovarian cancer adds to the understanding of how HT acts as a promoter of ovarian cancer carcinogenesis, as the results are in line with the current knowledge about HT-associated risks of cancers with similar epithelial origins. Because of this, it seems plausible that there is a causal association between HT and ovarian cancer. Other risk factors for ovarian cancer differ based on the type of tumor (mucinous vs. nonmucinous), supporting the hypothesis of different etiologies.[5, 6] The differences should be considered in research study design and suggest that different types of ovarian tumors should be viewed as separate diseases.

Strengths of Study

To our knowledge, our nationwide cohort study is the largest conducted thus far to explore the influence of HT on the risk of histologic subtypes of epithelial ovarian cancer. The validity of our outcome is considered to be high, as data from the Cancer Register validated the diagnoses (21–23). The agreement of histologic ovarian cancer diagnoses between the Pathology Register and the Cancer Register is high, and our estimates did not depend on the source of diagnoses.[24] The information on prescribed HT is transferred electronically from all Danish pharmacies by using bar codes, eliminating recall bias. Our information on both exposures and confounders was updated daily through the national registers, making it possible for us to account for changes in exposures. We excluded women with previous cancer because it might affect both the use of hormones and the subsequent risk of ovarian cancer. Our results were adjusted for age, time period, educational level, number of births, and history of hysterectomy, sterilization, unilateral oophorectomy, salpingo-oophorectomy, endometriosis, or infertility. There was, however, no significant confounding by any of the included variables. We found no evidence of earlier detection (surveillance bias) of ovarian tumors among women on HT. Finally, the stage of disease did not bias the differential association between HT and different tumor types.

Limitations of Study

Data from the National Register of Medicinal Product Statistics is not complete for the time period before January 1995. Thus, information about prescriptions for oral contraceptive use was not available for the women in current study who were 50 years of age or older from 1995−2005. Our incidence rate ratios may be underestimated because of confounding by use of oral contraceptives, as oral contraceptive use decreases the risk of ovarian cancer and often leads to HT.[25, 26] We were not able to restrict our analyses to nonobese women. The ovarian cancer risk associated with HT use is probably clearer in nonobese women (i.e., in women with a body mass index, measured as weight in kilograms divided by height in meters squared, <30).[27] Consequently, our results might be underestimated among nonobese. However, the Million Women Study adjusted data for oral contraceptive use, body mass index, age at menopause, alcohol consumption, smoking, and physical activity, and the adjustments did not result in material changes in their estimates.[3] Also, the NHS reported only minimal changes in the association between HT and the risk of ovarian cancer after adjustment for relevant potential confounders, including duration of oral contraceptive use, occurrence of natural menopause, and age at menarche.[12] The lack of information on family history of cancer might have caused an underestimation of risk in our results, as women with a family history of cancer are probably less likely to use hormones. Information on women who underwent surgical procedures was not available in the registers for the oldest women. Hysterectomy and oophorectomy reduce the risk of ovarian cancer and often lead to HT use, probably causing an underestimation of risk in older women in our results. However, despite our uneven adjustment for confounders, the risks for ovarian tumors were nearly identical across age groups.

9.6.5 Chemotherapy versus hormonal treatment in platinum- and paclitaxel-refractory ovarian cancer: a randomised trial of the German Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) Study Group Ovarian Cancer

  1. du Bois, W. Meier, H. J. Lück, G. Emons, V. Moebus, et al.
    Ann Oncol (2002) 13 (2): 251-257

The majority of patients with ovarian cancer are not cured by first-line treatment.Until now, no study could demonstrate any substantial benefit when exposing ovarian cancer patients to second-line chemotherapy. However, most treatment regimens induce toxicity, thus negatively influencing the quality of rather limited life spans. Here we evaluate whether a second-line chemotherapy can offer any benefit compared with a less toxic hormonal treatment. Patients and methods Patients with ovarian cancer progressing during platinum-paclitaxel containing first-line therapy or experiencing relapse within 6 months were eligible. Patients were stratified for response to primary treatment (progression versus no change/response), and measurable versus non-measurable disease. Treatment consisted of either treosulfan 7 g/m2infused over 30 min or leuprorelin 3.75 mg injected subcutaneously or intramuscularly. Both regimens were repeated every 4 weeks. Results This study began in late 1996, and after 2.5 years accrual an interim analysis was performed when several investigators reported their concern about a suspected lack of efficacy. Following this analysis the recruitment was stopped early and the 78 patients already enrolled were followed up. The majority of patients received treatment until progressive disease was diagnosed or death occurred. Treatment delay was observed rarely and dose reduction was performed only in the treosulfan arm in 5% of 150 courses. Overall, both treatment arms were well tolerated. No objective responses were observed. The median survival time was 36 and 30 weeks in the treosulfan and leuprorelin arms, respectively. Overall survival did not differ between patients with relapse 3–6 months after first-line chemotherapy compared with patients with progressive disease within 3 months.

Conclusions The selected patient population represents a subgroup with extremely poor prognosis. Accordingly, results were not impressive. Both treatment arms showed favourable toxicity data, but failed to show remarkable activity, thus adding only limited evidence to the issue of whether patients with refractory ovarian cancer might benefit from second-line chemotherapy. Even stratified analysis did not identify any subgroup of patients in whom the administration of second-line chemotherapy could demonstrate a clinically relevant survival benefit.

Despite the considerable progress that has been achieved in the treatment of advanced ovarian cancer during the last de-cades, the majority of patients are still not cured by first-line treatment. Therefore, development of effective second-line treatment strategies remains a clinically relevant issue. Today standard first-line regimens in many countries contain both paclitaxel and a platinum analogue (e.g. cisplatin [12] or carboplatin [35]). There are only limited data available reporting results gained from second-line therapy following failure of this new first-line regimen. Until now, no guidelines for the selection of second-line treatment regimens have reached universal acceptance [6]. Furthermore, the definitions of recurrent or relapsed disease according to the status of platinum resistance [7] were solely based on data from patients who did not receive the actual standard first-line regimens containing paclitaxel, and therefore have to be re-evaluated. The treatment-free interval, which offers a chance of gaining a benefit from re-treatment with paclitaxel and/or platinum, remains to be defined. However, patients progressing during or relapsing shortly after platinum-paclitaxel probably have a poor prognosis and can be regarded as refractory to both of the drugs they were exposed to. Until now, no study has demonstrated clearly any substantial benefit for these patients when treating them with second-line chemotherapy. However, most treatment regimens induce toxicity, thus negatively influencing the quality of rather limited life spans in this strictly palliative setting. Therefore, the AGO Study Group set about evaluating whether a second-line chemotherapy could offer any benefit compared with a less toxic hormonal treatment.

The decision to use an alkylating agent for second-line chemotherapy was based on the assumption that these agents, which had been part of first-line treatment of ovarian cancer for decades, could offer some benefit as second-line agent after removal from first-line regimens. Treosulfan (Ovastat®, medac, Germany) was chosen as alkylating agent because it has been registered and used frequently in older first-line regimens in Germany, due to a more favourable non-haematological toxicity profile compared with cyclophosphamide [89]. The published data for treosulfan as second-line treatment after platinum failure had been partially contradictory. Two studies using intravenous treosulfan reported response rates of up to 20% in 25 and 72 patients, respectively [1011]. The latter trial included 43 patients with platinum refractory ovarian cancer and showed a 21% response rate. Orally administered treosulfan resulted in response rates of 3, 14 and 19% in 30, 22 and 16 platinum pre-treated patients, respectively [1214]. The only study reporting results of oral treosulfan in platinum refractory patients observed only one response in 30 patients. Therefore, we decided to use intravenous treosulfan as standard chemotherapy arm in this trial.

Leuprorelin (leuproreline acetate; Enantone®, Takeda, Germany), a gonadotropin-releasing hormone (GnRH) analogue, was selected as hormonal treatment in the experimental arm of this study. It could be administered in a similar time schedule as the chemotherapy regimen (monthly injections) and had shown some activity in previously reported studies in platinum pre-treated ovarian cancer. In these trials, leuprorelin had been used either as single agent [1517] or in combination with megestrole acetate or tamoxifen [1819]. Overall, nine responses have been reported in 46 platinum pre-treated patients [cumulative odds ratio (OR) 19.6%; 95% confidence interval (CI) 9% to 34%]. A retrospective review reported higher efficacy for leuprorelin compared with goserelin, thus providing further support for selecting leuprorelin in favour of other GnRH analogues [17]. However, platinum resistance had been reported inconsistently in all these studies, thus leaving some questions unanswered regarding efficiency in this particular group of patients. Toxicity profiles of leuprorelin had been uniformely reported as being mild, making this option potentially useful in this strictly palliative setting. Tamoxifen, another hormonal treatment with an 11% overall response rate reported in a meta-analysis in recurrent ovarian cancer [20], was not selected for this study, because the study group felt that the different mode of application could hamper comparability.

The median observation period was 22.5 months for all patients. The early termination of recruitment resulted in a statistical power of 80% to detect a 20% survival difference (50% versus 69.9%) after 6 months with two-sided testing and an α error of 0.05.

Treatment and tolerability

The majority of patients received treatment until progressive disease was diagnosed or death occurred. The mean and median treatment periods, respectively, were 18 and 16 weeks in the treosulfan arm, and 13 and 10 weeks in the leuprorelin arm. Treatment delay was observed rarely and median intervals per course were 30.8 and 28.6 days in the treosulfan and leuprorelin arms, respectively. Dose reduction was performed only in the treosulfan arm in eight of 150 courses (5%) because of myelosuppression.

Overall, 150 chemotherapy courses and 122 hormonal treatment courses were evaluable for toxicity. Haematological toxicities higher than grade 2 were observed in only a few patients. Thrombocytopenia grade 3/4 occurred in four and one courses in the treosulfan and leuprorelin arms, respectively. Neutropenia grade 3/4 was only observed in one course in each arm and no infections or neutropenic fever was reported. Anaemia greater than grade 2 was observed after seven courses in the treosulfan arm and after two courses in the leuprorelin arm.

Non-haematological toxicities grade 3 or 4 were reported in only few patients in both arms. Treosulfan induced nausea and emesis in 9% of patients compared with 6% of patients in the leuprorelin arm. Hot flushes were reported by one patient in each arm. Three further patients in the treosulfan arm reported grade 3 pain (two patients) and neurotoxicity (one). The latter was due to remaining toxicity from prior platinum-paclitaxel. Alopecia was common but was due to prior treatment. Re-growth of patients’ hair took longer in the treosulfan arm than in the leuprorelin arm. About one-third of patients still had alopecia after treatment with treosulfan compared with 11% in the leuprorelin arm. Fatigue was reported more frequently in the chemotherapy arm (eight of 36 patients versus one of 37 patients, treosulfan versus leuprorelin;P = 0.014, Fisher’s exact test). Overall, both treatment arms were relatively well tolerated resulting in only one treatment cessation due to toxicity.


No objective responses were observed in either of the treatment arms. Disease stabilisation lasting at least 4 weeks (no change) was reported in nine and four patients in the chemotherapy and hormonal treatment arm, respectively. All but one patient showed progressive disease within a median observation period of 22 months. Median progression-free survival was 17 weeks for treosulfan and 10 weeks for leuprorelin (P = 0.035, Wilcoxon test). The difference between both treatment arms remained significant in favour of treosulfan after adjusting for treatment-free interval before study entry (P = 0.028). However, after 6 months only 23% and 14% of patients in the treosulfan and leuprorelin arms had not progressed; corresponding figures for the 12 month observation period were 9% and 5%, respectively (Figure 1).

View larger version:

Figure 1. Progression-free survival (median 17 and 10 weeks for treosulfan and leuprorelin, respectively; P<0.05, log rank test; Kaplan–Meier curves).

At the time of this analysis, seven patients in the treosulfan arm and eight patients in the leuprorelin arm are still alive with disease [hazard ratio (HR) 0.98; 95% CI 0.58–1.67]. The differences observed between the treatment arms did not reach statistical significance (P = 0.87, Wilcoxon test; Figure2). Again, adjusting for a treatment-free interval before study entry did not alter results. The median survival time was 36 and 30 weeks in the treosulfan and leuprorelin arm, respectively. About one-third of patients in each arm were alive after 12 months.

View larger version:

Figure 2. Survival (median 36 and 30 weeks for treosulfan and leuprorelin, respectively; P = 0.87, log rank test; Kaplan–Meier curves).

The impact of time to treatment failure after first-line chemo-therapy on second-line therapy efficacy was analysed bi-categorially. The cut-off was set at 13 weeks, thus comparing patients with progression under first-line therapy or early relapse within 3 months with patients who relapsed 3–6 months after completion of first-line chemotherapy. Overall, the difference between the groups with respect to progression-free survival did not reach statistical significance. Median progression-free survival was 11 and 12 weeks, respectively, for the two groups (P = 0.46, log rank test; HR 0.83; 95% CI 0.51–1.35). Furthermore, overall survival did not differ significantly between patients with relapse 3–6 months after first-line chemotherapy compared with patients with progressive disease within 3 months (P = 0.34, log rank test; HR 0.77; 95% CI 0.46–1.31). However, median survival was slightly superior in the group with a longer progression-free interval after first-line therapy (42 versus 25 weeks). The latter difference did not reach statistical significance and the Kaplan–Meier curves almost fell on top of each other shortly after 1 year of observation (data not shown).

The presence of bi-dimensionally measurable disease had a negative impact on treatment results. Patients with measurable disease showed a median progression-free survival of 11 weeks compared with 19 weeks in patients with non-measurable disease (P = 0.0006, log rank test). Again, overall survival was superior in the group of patients with non-measurable disease, but this difference did not reach statistical significance (median 47 versus 24 weeks; P = 0.18, log rank test). Only 29% of patients with measurable disease compared with 46% of patients with non-measurable disease were alive after 12 months (HR 1.93; 95% CI 0.73–5.16).

Subsequent treatment

In the treosulfan arm, 15 patients received third-line treatments, of whom three were changed over to leuprorelin. The remaining eight patients received: radiotherapy (one), tamoxifen (one) or chemotherapeutic drugs [topotecan (six), etoposide (one), liposomal doxorubicin (one), carboplatin (one), carboplatin-paclitaxel (one)]. Furthermore, 14 patients received fourth-line treatment, including tamoxifen (two), MPA (one), etoposide (two), topotecan (two), and one patient each idarubicin, gemcitabin or mitoxantrone i.p. Finally,three patients received fifth-line cyclophosphamide (one), etoposide (one) or radiotherapy (one). In the leuproreline arm, almost all patients received third-line therapy. Sixteen patients were crossed over to treosulfan, four received intraperitoneal mitoxantrone, two had liposomal doxorubicin and one patient each received etoposide, topotecan, carboplatin, paclitaxel-mitoxantrone or carboplatin-paclitaxel. Two patients received hormonal third-line treatment (one each received tamoxifen and MPA). Fourth-line treatment was offered to seven patients, including radiotherapy (one), topotecan (two), and one patient each liposomal doxorubicin-etoposide, etoposide or etoposide–5-fluorouracil (5-FU). Fifth-line treatment was offered to three patients, including paclitaxel, gemcitabin and 5-FU–platinum. The considerable use of third-line therapies after progression of disease might have hampered survival analysis, which in fact showed no significant difference between the treatment arms (although progression-free survival differed).

Previous SectionNext Section


This study represents a prospectively randomised trial in a very homogenous population. Only patients who were refractory to the standard first-line treatment of advanced ovarian cancer (i.e. platinum plus paclitaxel) were recruited. This selection represents a patient group with an extremely poor prognosis. At the moment, there is only limited evidence that these patients benefit from second-line chemotherapy at all, and more studies in this subgroup are necessary before any recommendations or guidelines can be established.

A randomised trial of the National Cancer Institute of Canada has shown an advantage for one arm over another when comparing 3-weekly topotecan days 1–5 to weekly topotecan in 78 patients, of whom 60% had received prior paclitaxel,and 60% were platinum refractory [23]. This advantage was limited to overall response (23% versus 8%). Progression-free survival differed only at a non-significant level (8 versus 13 weeks), and overall survival did not differ at all. Our trial showed a statistically significant advantage of one arm (treosulfan) with respect to progression-free survival, but failed to show any difference in overall survival. In addition, no differences with respect to response rates were observed. In fact, we did not observe any objective response. The latter could indicate a lack of activity of both study drugs, treosulfan and leuprorelin. However, even higher response rates as reported in the literature did not translate to longer progression-free and overall survival. A prospectively randomised trial comparing liposomal doxorubicin with topotecan included 254 platinum refractory patients; in addition, about two-thirds had received paclitaxel as part of prior therapy [24]. No significant differences were observed in the refractory subgroup: response rates were 7% and 12%, median progression-free survival was 9 and 14 weeks, and median survival was 33 and 37 weeks, respectively. Our observations of median progression free survival of 11 and 17 weeks and median survival of 30 and 36 weeks fit well with the reported data in this poor prognostic subgroup, although we did not observe any objective responses. Another randomised trial in 81 platinum refractory patients comparing paclitaxel with paclitaxel–epirubicin reported response rates of 17% and 34% translating to 2-year survival of 10% and 18% [25]. The corresponding 2-year survival in our trial was 19% and 22%, thus indicating the limited value of objective response rates as predictors for survival or progression-free survival in this poor prognostic subgroup of patients with truely refractory ovarian cancer.

Nevertheless, achieving an objective response might be beneficial in this palliative setting, especially if bulky tumours induce symptoms such as pain or bowl obstruction. However, objective response rates might not sufficiently reflect this potential benefit. Therefore, different response criteria that better reflect the palliative approach in these patients should be evaluated prospectively (e.g. symptom relief, reduction of pain medication or ability of enteral food intake). The development of better tools for the evaluation of genuine second-line chemotherapies becomes even more necessary when taking into account the fact that ovarian cancer becomes more of a chronic disease: mortality does not change substantially, but median and 5-year survival improves, thus indicating a growing need for efficient second-line and higher treatment. These therapies should allow tumour control and simultaneously should not reduce life quality.

This study reports mild toxicity data for both treatment arms, treosulfan and leuprorelin acetate, but, due to the very poor activity levels observed in both arms, adds only limited evidence to the issue of whether patients with refractory ovarian cancer benefit from second-line chemotherapy at all. Even stratified analysis in patients with progressive ovarian cancer versus patients experiencing relapse 3–6 months after first-line therapy, or patients with measurable versus non-measurable diseases, did not demonstrate any subgroup of patients in whom the administration of treosulfan second-line chemotherapy could demonstrate a clinically relevant benefit. Although a very short progression-free interval and the presence of bi-dimensionally measurable disease seemed to turn a bad prognosis into a worse prognosis, none of the differences between the strata showed a consistent and clinically relevant difference in survival. Only progression-free survival was influenced by these factors to some extent. Our data did not indicate that patients with a progression-free interval of >3 months but

However, results were disappointing in all subgroups. A rather small benefit was traded for a higher rate of fatigue in patients receiving chemotherapy. A gain of 6 weeks median progression-free survival in the superior chemotherapy arm in our study and some advantages with respect to response rates in other trials do not convincingly answer the question of whether second-line chemotherapy offers any benefit for patients with refractory ovarian cancer. Further studies are necessary to help to evaluate whether chemotherapy has a role in this subgroup of patients with a very unfavourable prognosis. A randomised comparison between best supportive care and the most active chemotherapy regimen available could theoretically be an appropriate design for such a trial. However, the German AGO investigators did not even broadly accept a randomisation between a hormonal treatment and a chemotherapy arm, as measured by an extremely slow recruitment rate. Furthermore, this study had to be closed prematurely after an interim analysis indicated only very limited activity in both treatment arms. A trial using best supportive care as one treatment arm would probably not be accepted either, although the above-mentioned efficacy data from chemotherapy studies do not provide more optimistic results.

Treosulfan showed an acceptable toxicity profile and at least some activity compared with leuprorelin acetate, thus allowing continuation of clinical research with this alkylating agent. Our subsequent trial in the refractory population compares treosulfan with topotecan (AGO protocol OVAR-2.3) and recruitment is much better, indicating that investigators more easily accept trials comparing two chemotherapy regimens. Quality of life evaluation was included in this protocol in an attempt to improve understanding of the nature of potential gains from second-line therapy.

Besides treosulfan and topotecan, which are further evaluated by our group, several chemotherapy agents have shown some activity in platinum- and paclitaxel-refractory ovarian cancer, and could serve as comparators in pending further trials: ifosfamide [26], hexamethylmelamine [27], gemcitabin [28] and liposomal doxorubicin [23,29]. The difficult task of recruiting large homogenous patient populations to second-line trials supports the ongoing discussions and activities in cooperative groups and networks, such as the worldwide Gynecologic Cancer InterGroup (GCIG), which is already planning and performing intergroup trials of second-line treatment of ovarian cancer.

Read Full Post »

Sex Hormones

Author: Larry H Bernstein, MD, FCAP

A steroid hormone is a steroid that acts as a hormone. Steroid hormones can be
grouped into five groups by the receptors to which they bind:

  • glucocorticoids,
  • mineralocorticoids,
  • androgens,
  • estrogens, and
  • progestogens.
  • Vitamin D derivatives, are a sixth closely related hormone system with homologous receptors. They have some of the characteristics of true steroids as receptor ligands.

Steroid hormones help control metabolism, inflammation, immune functions, salt
and water balance, development of sexual characteristics, and the ability to withstand
illness and injury. The term steroid describes both hormones produced by the body
and artificially produced medications that duplicate the action for the naturally occurring steroids

The natural steroid hormones are generally synthesized from cholesterol in the gonads and adrenal glands. These forms of hormones are lipids. They can pass through the cell membrane as they are fat-soluble,[4] and then bind to steroid hormone receptors (which may be nuclear or cytosolic depending on the steroid hormone) to bring about changes within the cell. Steroid hormones are generally carried in the blood, bound to specific carrier proteins such as sex hormone-binding globulin or corticosteroid-binding globulin. Further conversions and catabolism
occurs in the liver, in other “peripheral” tissues, and in the target tissues.

Synthetic steroids and sterols

A variety of synthetic steroids and sterols have also been contrived. Most are
steroids, but some non-steroidal molecules can interact with the steroid receptors
because of a similarity of shape. Some synthetic steroids are weaker or stronger
than the natural steroids whose receptors they activate.

Some examples of synthetic steroid hormones:
Glucocorticoids: alclometasone, prednisone, dexamethasone, triamcinolone
Mineralocorticoid: fludrocortisone
Vitamin D: dihydrotachysterol
Androgens: apoptone, oxandrolone, oxabolone, testosterone, nandrolone (also
known as anabolic steroids)
Estrogens: diethylstilbestrol (DES)
Progestins: danazol, norethindrone, medroxyprogesterone acetate,
17-Hydroxyprogesterone caproate.

Some steroid antagonists:
Androgen: cyproterone acetate
Progestins: mifepristone, gestrinone





The regulation of spermatogenesis by androgens

Lee B. Smith, William H. Walker
Seminars in Cell & Developmental Biology 30 (2014) 2–13

Testosterone is essential for maintaining spermatogenesis and male fertility.
However, the molecular mechanisms by which testosterone acts have not
begun to be revealed until recently. With the advances obtained from the use
of transgenic mice lacking or overexpressing the androgen receptor, the cell
specific targets of testosterone action as well as the genes and signaling pathways
that are regulated by testosterone are being identified. In this review, the critical
steps of spermatogenesis that are regulated by testosterone are discussed as well
as the intracellular signaling pathways by which testosterone acts. We also review
the functional information that has been obtained from the knock out of the androgen
receptor from specific cell types in the testis and the genes found to be regulated
after altering testosterone levels or androgen receptor expression.

The essence of female–male physiological dimorphism: Differential Ca2+-homeostasis
enabled by the interplay between farnesol-like endogenous sesquiterpenoids and
sex-steroids? The Calcigender paradigm

Arnold De Loof
General and Comparative Endocrinology 211 (2015) 131–146

Ca2+ is the most omnipresent pollutant on earth, in higher concentrations a real
threat to all living cells. When [Ca2+]i rises above 100 nM (=resting level), excess
Ca2+ needs to be confined in the SER and mitochondria, or extruded by the different
Ca2+-ATPases. The evolutionary origin of eggs and sperm cells has a crucial, yet
often overlooked link with Ca2+-homeostasis. Because there is no goal whatsoever
in evolution, gametes did neither originate ‘‘with the purpose’’ of generating a progeny
nor of increasing fitness by introducing meiosis. The explanation may simply be that
females ‘‘invented the trick’’ to extrude eggs from their body as an escape strategy
for getting rid of toxic excess Ca2+ resulting from a sex-hormone driven increased
influx into particular cells and tissues.
The production of Ca2+-rich milk, seminal fluid in males and all secreted proteins
by eukaryotic cells may be similarly explained. This view necessitates an upgrade
of the role of the RER-Golgi system in extruding Ca2+. In the context of insect
metamorphosis, it has recently been (re)discovered that (some isoforms of) Ca2+-
ATPases act as membrane receptors for some types of lipophilic ligands, in
particular for endogenous farnesol-like sesquiterpenoids (FLS) and, perhaps, for
some steroid hormones as well.
A novel paradigm, tentatively named ‘‘Calcigender’’ emerges. Its essence is: gender-
specific physiotypes ensue from differential Ca2+-homeostasis enabled by genetic
differences, farnesol/FLS and sex hormones. Apparently the body of reproducing
females gets temporarily more poisoned by Ca2+ than the male one, a selective
benefit rather than a disadvantage.

Sex differences in the expression of estrogen receptor alpha within noradrenergic
neurons in the sheep brain stem

J.L. Rose, A.S. Hamlin, C.J. Scott
Domestic Animal Endocrinology 49 (2014) 6–13

In female sheep, high levels of estrogen exert a positive feedback action
on gonadotropin releasing hormone (GnRH) secretion to stimulate a surge in
luteinizing hormone (LH) secretion. Part of this action appears to be via brain
stem noradrenergic neurons. By contrast, estrogen action in male sheep has
a negative feedback action to inhibit GnRH and LH secretion. To investigate
whether part of this sex difference is due to differences in estrogen action in
the brain stem, we tested the hypothesis that the distribution of estrogen
receptor a (ERα) within noradrenergic neurons in the brain stem differs
between rams and ewes. To determine the distribution of ERα, we used
double-label fluorescence immunohistochemistry for dopamine b-Hydroxylase,
as a marker for noradrenergic and adrenergic cells, and ERα. In the ventro-
lateral medulla (A1 region), most ERα-immunoreactive (-ir) cells were
located in the caudal part of the nucleus. Overall, there were more ERα-ir
cells in rams than ewes, but the proportion of double-labeled cells was did
not differ between sexes. Much greater numbers of ERα–ir cells were
found in the nucleus of the solitary tract (A2 region), but <10% were double
labeled and there were no sex differences. The majority of ERα-labeled cells
in this nucleus was located in the more rostral areas. Erα labeled cells were
found in several rostral brain stem regions but none of these were double
labeled and so were not quantified. Because there was no sex difference
in the number of ERα-ir cells in the brain stem that were noradrenergic,
the sex difference in the action of estrogen on gonadotropin secretion in
sheep is unlikely to involve actions on brain stem noradrenergic cells.

Androgens, estrogens, and second messengers

William Rosner, DJ Hryb, MS Khan, AM Nakhla, and NA Romas
Steroids 1998; 63:278 –281 PII S0039-128X(98)00017-8

Over the course of the last four decades, a detailed understanding of the
molecular mechanisms by which steroid hormones exert their effects has
evolved, and continues to evolve. The major focus of research in this area
has been on the manner in which steroid receptors activate transcription.
Pathways of steroid action other than by direct interaction with intracellular
receptors have received relatively little attention. However, there is a growing
body of evidence that steroid hormones exert effects through mechanisms
in addition to those involving their classic intracellular receptors. One such
mechanism is based on the observation that a number of cells have receptors
on their plasma membranes for the plasma protein, sex hormone binding
globulin (SHBG). It is the purpose of this review to briefly describe our current
knowledge of this system.

SHBG binds to a receptor (RSHBG) on cell membranes cAMP and the steroid-SHBG-RSHBG system
Biology of the SHBG-RSHBG system

Relation between the affinity of steroid for SHBG and its potency in inhibiting
the binding of SHBG to RSHBG.

KA (SHBG) = Association constant for SHBG and the indicated steroid.
Ki SHBG-RSHBG = The inhibition constant for the indicated steroid on the
binding of SHBG to RSHBG.

PSA secretion was stimulated by DHT. Although estradiol alone had no effect
on PSA secretion, it caused an increase equal to that seen with DHT if the
prostate tissue was first loaded with SHBG, e.g., if RSHBG was occupied by
SHBG. Because estradiol-SHBG increases intracellular cAMP, we ascertained
whether other compounds that raise cAMP (forskolin), or cAMP itself, could
increase PSA secretion. Such was the case. cAMP begins its signal cascade
by activating protein kinase A (PKA) so that if estradiol-SHBG increases PSA
secretion by a mechanism involving cAMP, inhibition of PKA should block
estradiol-SHBG-initiated PSA secretion. Estradiol-SHBG failed to stimulate
PSA when PKA was inhibited with PKI. On the other hand, DHT-stimulated
PSA secretion, which does not involve PKA, was not inhibited by PKI. That
the effect of estradiol-SHBG was independent of the estrogen receptor was
shown by the lack of inhibition of estrogen-stimulated PSA secretion by two
anti-estrogens, tamoxifen and ICI 164,284. The promoter of the PSA gene
has an androgen response element, and both PSA secretion and the
expression of PSA mRNA are androgen-regulated. We investigated the
effect of hydroxyflutamide and cyproterone acetate. Both potent anti-
androgens, on the E2-SHBG-mediated increase in PSA secretion. secretion.
They also blocked the effect of E2-SHBG on PSA secretion. Since E2 is
not exerting its effect by binding to the AR, e.g., it is not its cognate ligand,
the E2-induced secretion of PSA observed in this study reflects ligand-
independent activation of the AR.26 Thus, estradiol activates a typical
AR-mediated event, PSA synthesis and secretion, by activating SHBG-
RSHBG. These data make clear the fact that there is cross-talk between a
steroid hormone-engendered event at the cell membrane and a classic
intracellular steroid hormone receptor.
Abbreviations: PSA, prostate specific antigen; DHT, dihydrotestosterone;
E2, estradiol; PKI, inhibitor of protein kinase A; ICI 164,384 (a pure anti-
estrogen); 2MeOE2, 2 methoxyestradiol; Cypro, cyproterone acetate,
OHFlut, hydroxyflutamide.

Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte
maturation by endogenous estrogens in zebrafish

Yefei Pang, Peter Thomas
Developmental Biology 342 (2010) 194–206

Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly
known as GPR30) were investigated in zebrafish. Estradiol-17β (E2) and G-1,
a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting
the presence of GPER which was confirmed by immunocytochemistry using
a specific GPER antibody. Incubation of follicle-enclosed oocytes with an
aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian
follicle cell layers significantly increased spontaneous OM which was partially
reversed by co-treatment with either 100 nM E2 or G-1. Incubation of
denuded oocytes with the GPER antibody blocked the inhibitory effects of
estrogens on OM, whereas microinjection of estrogen receptor alpha (ERα)
antisense oligonucleotides into the oocytes was ineffective. The results
suggest that endogenous estrogens produced by the follicle cells inhibit or
delay spontaneous maturation of zebrafish oocytes and that this estrogen
action is mediated through GPER. Treatment with E2 and G-1 also attenuated
the stimulatory effect of the teleost maturation-inducing steroid, 17,20 β-
dihyroxy-4-pregnen-3-one (DHP), on OM.  Moreover, E2 and G-1 down-
regulated the expression of membrane progestin receptor alpha (mPRα),
the intermediary in DHP induction of OM. Conversely DHP treatment caused
a N50% decline in GPER mRNA levels. The results suggest that estrogens
and GPER are critical components of the endocrine system controlling
the onset of OM in zebrafish. A model is proposed for the dual control of the
onset of oocyte maturation in teleosts by estrogens and progestins acting
through GPER and mPRα, respectively, at different stages of oocyte
Reprint of ’’GPR30 mediates estrogen rapid signaling and neuroprotection’’

Hui Tang, Q Zhang, L Yang, Y Dong, M Khan, F Yang, DW Brann, R Wang
Molecular and Cellular Endocrinology 389 (2014) 92–98

G-protein-coupled estrogen receptor-30 (GPR30), also known as G-protein
estrogen receptor-1 (GPER1), is a putative extranuclear estrogen receptor
whose precise functions in the brain are poorly understood. Studies using
exogenous administration of the GPR30 agonist, G1 suggests that GPR30
may have a neuroprotective role in cerebral ischemia. However, the
physiological role of GPR30 in mediating estrogen (E2)-induced neuro-
protection in cerebral ischemia remains unclear. Also unclear is whether
GPR30 has a role in mediating rapid signaling by E2 after cerebral ischemia,
which is thought to underlie its neuroprotective actions. To address these
deficits in our knowledge, the current study examined the effect of antisense
oligonucleotide (AS) knockdown of GPR30 in the hippocampal CA1 region
upon E2-BSA induced neuroprotection and rapid kinase signaling in a rat
model of global cerebral ischemia (GCI). Immunohistochemistry demonstrated
that GPR30 is strongly expressed in the hippocampal CA1 region and
dentate gyrus, with less expression in the CA3 region. E2-BSA exerted
robust neuroprotection of hippocampal CA1 neurons against GCI, an effect
abrogated by AS knockdown of GPR30. Missense control oligonucleotides had
no effect upon E2-BSA-induced neuroprotection, indicating specificity of the
effect. The GPR30 agonist, G1 also exerted significant neuroprotection against
GCI. E2-BSA and G1 also rapidly enhanced activation of the prosurvival
kinases, Akt and ERK, while decreasing proapototic JNK activation. Importantly,
AS knockdown of GPR30 markedly attenuated these rapid kinase signaling
effects of E2-BSA. As a whole, the studies provide evidence of an important
role of GPR30 in mediating the rapid signaling and neuroprotective actions
of E2 in the hippocampus.
Regulation of brain microglia by female gonadal steroids

Pardes Habib, Cordian Beyer
Journal of Steroid Biochemistry & Molecular Biology 2015; 146: 3–14

Microglial cells are the primary mediators of the CNS immune defense system
and crucial for shaping inflammatory responses. They represent a highly
dynamic cell population which is constantly moving and surveying their
environment. Acute brain damage causes a local attraction and activation of
this  immune cell type which involves neuron-to-glia and glia-to-glia interactions.
The prevailing view attributes microglia a “negative” role such as defense and
debris elimination. More topical studies also suggest a protective and “positive”
regulatory function. Estrogens and progestins exert anti-inflammatory and
neuroprotective effects in the CNS in acute and chronic brain diseases.
Recent work revealed that microglial cells express subsets of classical and
non-classical estrogen and progesterone receptors in a highly dynamic way.
In this review article, we would like to stress the importance of microglia for
the spreading of neural damage during hypoxia, their susceptibility to functional
modulation by sex steroids, the potency of sex hormones to switch microglia
from a pro-inflammatory M1 to neuroprotective M2 phenotype, and the
regulation of pro-and anti-inflammatory properties including the inflammasome.
We will further discuss the possibility that the neuroprotective action of sex
steroids in the brain involves an early and direct modulation of local microglia
cell function. Neuroprotection by gonadal steroid hormones in acute brain
damage requires cooperation with astroglia and microglia

Sonja Johann, Cordian Beyer

The neuroactive steroids 17β-estradiol and progesterone control a broad
spectrum of neural functions. Besides their roles in the regulation of classical
neuroendocrine loops, they strongly influence motor and cognitive systems,
behavior, and modulate brain performance at almost every level. Such a
statement is underpinned by the widespread and lifelong expression pattern
of all types of classical and non-classical estrogen and progesterone receptors
in the CNS. The life-sustaining power of neurosteroids for tattered or seriously
damaged neurons aroused interest in the scientific community in the past years
to study their ability for therapeutic use under neuropathological challenges.
Documented by excellent studies either performed in vitro or in adequate animal
models mimicking acute toxic or chronic neuro-degenerative brain disorders,
both hormones revealed a high potency to protect neurons from damage
and saved neural systems from collapse. Unfortunately, neurons, astroglia,
microglia, and oligodendrocytes are comparably target cells for both steroid
hormones. This hampers the precise assignment and understanding of
neuroprotective cellular mechanisms activated by both steroids. In this article,
we strive for a better comprehension of the mutual reaction between these
steroid hormones and the two major glial cell types involved in the maintenance
of brain homeostasis, astroglia and microglia, during acute traumatic brain
injuries such as stroke and hypoxia. In particular, we attempt to summarize
steroid-activated cellular signaling pathways and molecular responses in these
cells and their contribution to dampening neuroinflammation and neural

Photoperiod influences the ontogenetic expression of aromatase
and estrogen receptor α in the developing tilapia brain.

Li-Hsueh Wang, Ching-Lin Tsai
General and Comparative Endocrinology 2006; 145: 62–66

Neural development is determined not only by genetic regulation, but also
by environmental cues. Central estrogen-forming/estrogen-sensitive systems
play an important role in the neural development of the brain. In the present
study, the quantitative reverse transcription-polymerase chain reaction method
was used to investigate the effects of photoperiod on the ontogenetic
expression of aromatase and estrogen receptor a (ERα) in the developing
tilapia brain. Before day 5 post-hatch, brain aromatase mRNA expression was
significantly decreased by constant light but not influenced by constant darkness.
During this period, brain ERα mRNA expression was significantly increased
under both constant light and constant darkness. Between days 5 and 10, and
between days 10 and 15, neither brain aromatase nor brain ERα expression
was altered under constant darkness and constant light. These results indicate
that the ontogenetic expression of brain aromatase and brain ERα is not via a
light-inducing process but influenced by a light-entraining signal during the
very early period of development.

Orphanin FQ-ORL-1 Regulation of Reproduction and Reproductive Behavior in
the Female

Kevin Sinchak, Lauren Dalhousay, Nayna Sanathara
Vitamins and Hormones 187-220.

Orphanin FQ (OFQ/N) and its receptor, opioid receptor-like receptor-1 (ORL-1),
are expressed throughout steroid-responsive limbic and hypothalamic circuits
that regulate female ovarian hormone feedback and reproductive behavior
circuits. The arcuate nucleus of the hypothalamus (ARH) is a brain region
that expresses OFQ/N and ORL-1 important for both sexual behavior and
modulating estradiol feedback loops. Within the ARH, the activation of the
OFQ/N-ORL-1 system facilitates sexual receptivity (lordosis) through the
inhibition of β-endorphin neuronal activity. Estradiol initially activates ARH
β-endorphin neurons to inhibit lordosis. Simultaneously, estradiol upregulates
coexpression of OFQ/N and progesterone receptors and ORL-1 in ARH
β-endorphin neurons. Ovarian hormones regulate pre- and postsynaptic
coupling of ORL-1 to its G protein-coupled signaling pathways. When the
steroid-primed rat is nonreceptive, estradiol acts pre- and postsynaptically
to decrease the ability of the OFQ/N-ORL-1 system to inhibit ARH β-endorphin
neurotransmission. Conversely, when sexually receptive, ORL-1 signaling is
restored to inhibit β-endorphin neurotransmission. Although steroid signaling
that facilitates lordosis converges to deactivate ARH.
Estradiol Activates the Prostate Androgen Receptor and Prostate specific Antigen
Secretion through the Intermediacy of Sex Hormone-binding Globulin

Atif M. Nakhla, Nicholas A. Romas, and William Rosner
J Biol Chem Mar 14, 1997; 272(11): 6838–6841

These experiments were designed to examine the relationship between the
effects of steroid hormones mediated by classic intracellular steroid hormone
receptors and those mediated by a signaling system subserved at the plasma
membrane by a receptor for sex hormone binding globulin. It is known that
unliganded sex hormone-binding globulin (SHBG) binds to a receptor (RSHBG)
on prostate membranes. The RSHBG*SHBG complex is rapidly activated by
estradiol to stimulate adenylate cyclase, with a resultant increase in intracellular
cAMP. In this paper we examine the effect of this system on a prostate gene
product known to be activated by androgens, prostate-specific antigen.
We have shown previously that estradiol (E2) participates in a signaling
system that originates, not within the cell, but at the plasma membrane.
Through the intermediacy of the plasma protein, sex hormone-binding
globulin (SHBG), it causes the generation of cAMP. In brief, unliganded
SHBG binds to a receptor (RSHBG) on certain cell surfaces and the
RSHBG*SHBG complex is rapidly activated by E2 to stimulate adenylate cyclase,
with a resultant increase in intracellular cAMP. There is a paucity of information
on events subsequent to the generation of cAMP by this system. In this paper
we examine the effect of E2-SHBG-RSHBG on an androgen responsive gene.
The gene for prostate-specific antigen (PSA) contains an androgen response
element. After binding its cognate ligand, the androgen receptor (AR) interacts
with this response element to initiate PSA mRNA transcription and secretion.
We show that, in the absence of androgens, E2 in concert with SHBG*RSHBG,
acts at the cell membrane to cause secretion of PSA and that this effect is
blocked by anti-androgens. This observation provides a first functional link
between a classic steroid hormone receptor and a cell membrane-mediated
steroidal effect. In serum-free organ culture of human prostates,
dihydrotestosterone caused an increase in prostate specific antigen secretion.
This event was blocked by the anti-androgens cyproterone acetate and
hydroxyflutamide. In the absence of androgens, estradiol added to prostate
tissue, whose RSHBG was occupied by SHBG, reproduced the results seen
with dihydrotestosterone. Neither estradiol alone nor SHBG alone duplicated
these effects. The estradiol*SHBG-induced increase in prostate-specific
antigen was not blocked by anti-estrogens, but was blocked both by anti-
androgens and a steroid (2-methoxyestradiol) that prevents the binding of
estradiol to SHBG. Furthermore, an inhibitor of protein kinase A prevented
the estradiol*SHBG-induced increase in prostate-specific antigen but not
that which followed dihydrotestosterone. These data indicate that there is a
signaling system that amalgamates steroid-initiated intracellular events
with steroid-dependent occurrences generated at the cell membrane and
that the latter signaling system proceeds by a pathway that involves protein
kinase A.
Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid
hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85

Thyroid hormones (THs) are well-known regulators of development and
metabolism in vertebrates. There is increasing evidence that THs are also
involved in gonadal differentiation and reproductive function. Changes in TH
status affect sex ratios in developing fish and frogs and reproduction
(e.g., fertility), hormone levels, and gonad morphology in adults of species of
different vertebrates. In this review, we have summarized and compared the
evidence for cross-talk between the steroid hormone and thyroid axes and
present a comparative model. We gave special attention to TH regulation of
sex steroid synthesis and action in both the brain and gonad, since these are
important for gonad development and brain sexual differentiation and have
been studied in many species. We also reviewed research showing that
there is a TH system, including receptors and enzymes, in the brains and
gonads in developing and adult vertebrates. Our analysis shows that THs
influences sex steroid hormone synthesis in vertebrates, ranging from fish
to pigs. This concept of crosstalk and conserved hormone interaction has
implications for our understanding of the role of THs in reproduction, and
how these processes may be dysregulated by environmental endocrine
Inverse relationship between hSHBG affinity for testosterone and hSHBG
concentration revealed by surface plasmon resonance

Laurence Heinrich-Balard, Wael Zeinyeh, Henri Déchaud, Pascaline Rivory, et al.
Molecular and Cellular Endocrinology 399 (2015) 201–207

A wide range of human sex hormone-binding globulin (hSHBG) affinity constants
for testosterone (KA_hSHBG) has been reported in literature. To bring new insight
on the KA_hSHBG value, we implemented a study of the molecular interactions
occurring between testosterone and its plasma transport proteins by using
surface plasmon resonance. The immobilization on the sensor-chip of a
testosterone derivative was performed by an oligoethylene glycol linker.
For different plasmas with hSHBG concentrations, an assessment of the
KA_hSHBG was obtained from a set of sensor-grams and curve-fitting these
data.We observed that KA_hSHBG decreased, from at least two decades,
when the plasma hSHBG concentration increased from 4.4 to 680 nmol/L.
Our study shows a wide biological variability of KA_hSHBG that is related
to the hSHBG concentration.
These unexpected results may have a physiological significance and question
the validity of current methods that are recommended for calculating free
testosterone concentrations to evaluate androgen disorders in humans.
Intracrinology in action: Importance of extragonadal sex steroid biosynthesis
and inactivation in peripheral tissues in both women and men.

Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 131–132

It seems appropriate, as introduction, to summarize the mechanisms at the
basis of the new paradigm of steroid biosynthesis in the human peripheral
tissues, namely intracrinology. While the first clinical proof of the role of
extragonadal sex steroid biosynthesis was obtained with combined androgen
blockade in men treated for prostate cancer, the first demonstration of the
efficacy of DHEA replacement therapy was on the symptoms of vulvovaginal
atrophy in postmenopausal women; (Archer, this issue).
DHEA is transformed specifically in each cell of each peripheral tissue into
the proper amounts of estrogens and/or androgens, depending upon the
local expression of the appropriate steroid forming enzymes; (Labrie, this issue).
Most importantly, the sex steroids synthesized and acting intracellularly in
peripheral tissues are also inactivated locally before being released in the
extracellular space, thus maintaining the serum levels of estradiol and
testosterone at biologically inactive concentrations, thus avoiding systemic
exposure to sex steroids during menopause as well illustrated by atrophy
of the endometrium.
As mentioned above, that extragonadal androgen biosynthesis is clinically
important became obvious in 1982 when the addition of the antiandrogen
flutamide to castration provided very exciting and unexpected beneficial results
(Labrie, this issue). In fact, combining a pure anti-androgen to castration has
been the first treatment shown to prolong life in prostate cancer and very clearly
confirmed by the prolongation of life of 2.2–4.8 months observed following
addition of MDV-3100 or abiraterone to castration resistant prostate cancer
patients (Grist et al., this issue). (Mizokami et al., this issue) very competently
complement the mechanisms potentially involved in extragonadal steroid
biosynthesis. A repeated observation is the association between serum DHEA
levels and increased longevity, a subject reviewed by Ohlsson et al., this issue.
Most importantly, a subject which remains to be supported by long-term clinical
trials but which shows very promising preclinical data is the possibility of a
beneficial effect of DHEA on brain functions, especially cognition, memory
and delayed development of mild cognitive impairment and Alzheimer’s
disease (see Starka et al.; Soma et al; Pluchino et al; Maggio et al.; Hill et al.,
this issue). The information summarized in the very up-to-date manuscripts
of this special JSBMB issue has the potential of opening the way to a prodrug
replacement therapy already well illustrated on the symptoms and signs of
vulvovaginal atrophy and sexual dysfunction (Archer, this issue). The
administration to sex steroid deficient women of an appropriate amount of
DHEA able to correct the symptoms of vulvovaginal atrophy (mostly estrogen-
sensitive) and sexual dysfunction (androgen-sensitive), and potentially, in the
future, other problems of menopause, does permit to the sex steroid-deficient
women to benefit from a normal/sufficient level of sex steroids in specific tissues
using the enzymes developed over 500 million years to permit a better quality
of life during the menopausal years.

Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans

Alain Belanger, Georges Pelletier, Fernand Labrie, Olivier Barbier and Sarah Chouinard
TRENDS in Endocrinology and Metabolism 2003; 14(10):473-78

In humans, 3b-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD and
5α-reductase activities in androgen target tissues, such as the prostate and
skin, convert dehydroepiandrosterone, androstenedione and testosterone into
the most potent natural androgen dihydrotestosterone (DHT). This androgen
is converted mainly in situ into two phase I metabolites, androsterone (ADT)
and androstane-3α,17β-diol (3α-DIOL), which might be back converted to DHT.
Here, we discuss the recent findings regarding the characterization of specific
UDP glucuronosyltransferases (UGTs), UGT2B7, B15 and B17, responsible for
the glucuronidation of these metabolites. The tissue distribution and cellular
localization of the UGT2B transcripts and proteins in humans clearly indicate
that these enzymes are synthesized in androgen-sensitive tissues. It is
postulated that the conjugating activity of UGT enzymes is the main mechanism
for modulating the action of steroids and protecting the androgen-sensitive
tissues from deleteriously high concentrations of DHT, ADT and 3α-DIOL.
Synthesis and Evaluation of Potential Radioligands for the Progesterone Receptor

R.M. Hoyte, W. Rosner, I.S. Johnson, J. Zielinski, and R. B. Hochberg
J. Med. Chem. 1985; 28: 1695-1699

Several steroidal analogues were synthesized as potential y-emitting radioligands
for the progesterone receptor. Each of these compounds was tested as an inhibitor
of the specific binding of [3H]-17α,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione
(R5020) to the progesterone receptor in rabbit uterine cytosol. R5020 is a well-
known progestin with high affinity for the receptor. Of the compounds synthesized,
aromatic N-substituted (2-17 steroidal carboxamides inhibited the binding only
poorly. Three compounds, 16α-iodo-4-estren-17β-ol-3-one, 17α-[2(E)-iodovinyl]
-4-estren-17β-ol-3-one, and 17α-[2(Z)-iodovinyl]-4-estren-l7β-ol-3-one are
excellent competitors, each having a Ki less than or equal to that of the natural
progestin, progesterone. Since similar iodinated analogues of estrogens
have been shown to be extremely stable both in vivo and in vitro, these compounds
are potentially useful ligands for the progesterone receptor.

Estradiol concentration and the expression of estrogen receptors in the testes of
the domestic goose (Anser anser f. domestica) during the annual reproductive cycle

Leska, J. Kiezun, B. Kaminska, L. Dusza
Domestic Animal Endocrinology 51 (2015) 96–104

Seasonal fluctuations in the activity of bird testes are regulated by a complex mechanism
where androgens play a key role. Until recently, the role played by estrogens in males has
been significantly underestimated. However, there is growing evidence that the proper
functioning of the testes is associated with optimal estradiol (E2) concentration
in both the plasma and testes of many mammalian species. Estrogens are
gradually emerging as very important players in hormonal regulation of
reproductive processes in male mammals. Despite the previously mentioned,
it should be noted that estrogenic action is limited by the availability of
specific receptors – estrogen receptor alpha (ERα) and estrogen receptor beta
(ERβ). Interestingly, there is a general scarcity of information concerning the
estrogen responsive system in the testes of male birds, which is of particular
interest in exploring the phenomenon of seasonality of reproduction. To address
this question, we have investigated for the first time the simultaneous
expression of testicular ERα and ERβ genes and proteins with the
accompanying plasma and testicular E2 concentrations during the annual
reproductive cycle of male bird. The research model was the domestic
goose (Anser anser f. domestica), a species whose annual reproductive
cycle can be divided into 3 distinct phases characterized by changes
in testicular activity. It has been revealed that the stable plasma E2 profile
did not correspond to changing intratesticular E2 profile throughout the
experiment. The expression of ERα and ERβ genes and proteins was detected
in gander testes and it fluctuated on a seasonal basis with lower level in
breeding and sexual reactivation stages and higher level during the
nonbreeding stage. Our results demonstrated changes in testicular sensitivity
to estrogens in male domestic goose during the annual reproductive cycle.
The seasonal pattern of estrogen receptors (ERs) expression was analyzed
against the hormonal background and a potential mechanism of ERs regulation
in bird testes was proposed. The present study revealed seasonal variations
in the estrogen responsive system, but further research is needed to fully
explore the role of estrogens in the reproductive tract of male birds.

Effects of 5α-dihydrotestosterone on expression of genes related to steroidogenesis
and spermatogenesis during the sex determination and differentiation periods of
the pejerrey, Odontesthes bonariensis

Anelisa González, Juan I. Fernandino, Gustavo M. Somoza
Comparative Biochemistry and Physiology, Part A 182 (2015) 1–7

Sex steroid hormones are important players in the control of sex differentiation
by regulating gonadal development in teleosts. Although estrogens are clearly
associated with the ovarian differentiation in teleosts, the effects of androgens
on early gonadal development are still a matter of debate. Traditionally,
11-ketotestosterone (11-KT) is considered themajor androgen in fish; however,
5α-dihydrotestosterone (5α-DHT), the most potent androgen in tetrapods, was
recently found in fish testis and plasma, but its physiological role is still unknown.
In this context, the expression of genes associated with steroidogenesis and
spermatogenesis, body growth and sex differentiation were assessed in
Odontesthes bonariensis larvae fed with food supplemented with two doses of
5α-DHT (0.1 and 10 μg/g of food) from hatching to 6 weeks of age. At the lowest
dose, 5α-DHT treated larvae showed an estrogenic gene expression pattern, with
low hsd11β2 and high cyp19α1α and er2 expression levels with no differences
in sex ratio. At the highest dose, 5α-DHT produced a male-shifted sex ratio and
the larvae exhibited a gene expression profile characteristic of an advancement
of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No
differences were observed in somatic growth. These results suggest that in
this species, 5α-DHT could have a role on sex differentiation and its effects
can differ according to the dose.
Do androgens link morphology and behavior to produce phenotype-specific
behavioral strategies?

Douglas G. Barron, Michael S. Webster, Hubert Schwabl
Animal Behaviour 100 (2015) 116e124

Morphological and behavioral traits often covary with each other, and the links
between them may arise from shared physiological mechanisms. In particular,
androgens such as testosterone have emerged as prime candidates for linking
behaviour and morphology due to the environmental sensitivity and pleiotropic
effects of these hormones. In this study we investigated the hypothesis that
androgens simultaneously relate to morphological and behavioral variation,
thereby producing the integrated reproductive phenotypes of male red-backed
fairy-wrens, Malurus melanocephalus. Males of this species can adopt one of
three discrete breeding phenotypes: breeding in red/black plumage, breeding
in brown plumage, or remaining as nonbreeding brown natal auxiliaries. Although
the expression of morphological traits in this species is regulated by androgens
and phenotypes differ in baseline androgen levels (red/black breeder > brown
breeder > auxiliary), injection with GnRH failed to expose phenotype specific
constraints on androgen production. Observations of territoriality, nestling
feeding and extraterritorial forays revealed phenotype-specific patterns of mating
and parental effort, yet these were largely related to age and were not correlated
with baseline or GnRH-induced androgen levels, or the androgen change between
these points. While these findings support the idea that morphological and
behavioral traits are linked via phenotypic correlations, they do not support
the hypothesis that behavioral differences arise from variation in circulating
androgens or the capacity to produce them.
Effects of sex steroids on expression of genes regulating growth-related
mechanisms in rainbow trout (Oncorhynchus mykiss)

Beth M. Cleveland, Gregory M. Weber
General and Comparative Endocrinology xxx (2015) xxx–xxx

Effects of a single injection of 17b-estradiol (E2), testosterone (T), or
5b-dihydrotestosterone (DHT) on expression of genes central to the
growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle
regulatory factors, transforming growth factor-beta (TGFβ) superfamily
signaling cascade, and estrogen receptors were determined in rainbow
trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in
addition to regulating GH sensitivity and IGF production, sex
steroids also affected expression of IGF binding proteins, as E2, T,
and DHT increased expression of igfbp2β and E2 also increased
expression of igfbp2 and igfbp4. Regulation of this system also occurred
in white muscle in which E2 increased expression of igf1, igf2, and
igfbp5β1, suggesting anabolic capacity may be maintained in white
muscle in the presence of E2. In contrast, DHT decreased expression
of igfbp5β1. DHT and T decreased expression of myogenin, while other
muscle regulatory factors were either not affected or responded similarly
for all steroid treatments. Genes within the TGFβ superfamily signaling
cascade responded to steroid treatment in both liver and muscle,
suggesting a regulatory role for sex steroids in the ability to transmit
signals initiated by TGFβ superfamily ligands, with a greater number
of genes responding in liver than in muscle. Estrogen receptors were
also regulated by sex steroids, with era1 expression increasing for all
treatments in muscle, but only E2- and T-treatment in liver. E2 reduced
expression of erb2 in liver. Collectively, these data identify how
physiological mechanisms are regulated by sex steroids in a manner
that promotes the disparate effects of androgens and estrogens on
growth in salmonids.
Distribution and function of 3′,5′-Cyclic-AMP phosphodiesterases in the human ovary

T.S. Petersen, S.G. Kristensen, J.V. Jeppesen, .., K.T. Macklon, C.Y. Andersen
Molecular and Cellular Endocrinology 403 (2015) 10–20

The concentration of the important second messenger cAMP is regulated by
phosphodiesterases (PDEs) and hence an attractive drug target. However,
limited human data are available about the PDEs in the ovary. The aim of the
present study was to describe and characterise the PDEs in the human ovary.
Results were obtained by analysis of mRNA microarray data from follicles and
granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs,
immunohisto-chemical analysis of ovarian sections and by studying the effect
of PDE inhibitors on progesterone production from cultured GCs. We found that
PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A
was not detected. PDE8B was differentially expressed during folliculogenesis.
In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone
secretion while PDE4 inhibition increased forskolin-stimulated progesterone
secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as
the major PDEs in the human ovary.
Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while
potently inhibiting estradiol-3-O-glucuronidation

Liangliang Zhu, Ling Xiao, Yangliu Xia, .., Yan Wu, Ganlin Wu, Ling Yang
Toxicology and Applied Pharmacology 283 (2015) 109–116

This in vitro study investigates the effects of diethylstilbestrol (DES), a widely
used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O)
glucuronidation, via culturing human liver microsomes (HLMs) or
recombinant UDP-glucuronosyl-transferases (UGTs) with DES and E2.
DES can potently inhibit E2-3-O-glucuronid-ation in HLM, a probe reaction
for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive
inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than
the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation,
the acceleration is observed on E2-17-O-glucuronidation in HLM, in which
cholestatic E2-17-O-glucuronide is generated. In the presence of DES
(0–6.25 μM), Km values for E2-17-Oglucuronidation are located in the
range of 7.2–7.4 μM, while Vmax values range from 0.38 to 1.54 nmol/min/mg.
The mechanism behind the activation in HLM is further demonstrated by
the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing
E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate Vmax from
0.016 to 0.81 nmol/min/mg, while lifting Km in a much lesser extent from 4.4 to
11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding
site model, with KA, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56,
respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation
are not observed in liver microsomes from several common experimental animals.
In summary, this study issues new potential toxic mechanisms for DES: potently
inhibiting the activity of UGT1A1 and powerfully accelerating the formation of
cholestatic E2-17-O-glucuronide by UGT1A4.
Dehydroepiandrosterone: A neuroactive steroid

Luboslav Stárka, Michaela Dusková, Martin Hill
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 254–260

Dehydroepiandrosterone (DHEA) and its sulfate bound form (DHEAS) are important
steroids of mainly adrenal origin. They are produced also in gonads and in the brain.
Dehydroepiandrosterone easily crosses the brain–blood barrier and in part is also
produced locally in the brain tissue. In the brain, DHEA exerts its effects after
conversion to either testosterone and dihydrotestosterone or estradiol via androgen
and estrogen receptors present in the most parts of the human brain, through
mainly non-genomic mechanisms, or eventually indirectly via the effects of its
metabolites formed locally in the brain. As a neuroactive hormone, DHEA in
cooperation with other hormones and transmitters significantly affects some
aspects of human mood, and modifies some features of human emotions and
behavior. It has been reported that its administration can increase feelings of well-
being and is useful in ameliorating atypical depressive disorders. It has
neuroprotective and antiglucocorticoid activity and modifies immune reactions,
and some authors have also reported its role in degenerative brain diseases.
Here we present a short overview of the possible actions of dehydroepiandrosterone
and its sulfate in the brain, calling attention to various mechanisms of their action
as neurosteroids and to prospects for the knowledge of their role in brain disorders.
Androgens and mammalian male reproductive tract development

Aki Murashima, Satoshi Kishigami, Axel Thomson, Gen Yamada
Biochimica et Biophysica Acta 1849 (2015) 163–170

One of the main functions of androgen is in the sexually dimorphic development of
the male reproductive tissues. During embryogenesis, androgen determines the
morphogenesis of male specific organs, such as the epididymis, seminal vesicle,
prostate and penis. Despite the critical function of androgens in masculinization,
the downstream molecular mechanisms of androgen signaling are poorly
understood. Tissue recombination experiments and tissue specific androgen
receptor (AR) knockout mouse studies have revealed epithelial or mesenchymal
specific androgen-AR signaling functions. These findings also indicate that
epithelial–mesenchymal interactions are a key feature of AR specific activity,
and paracrine growth factor action may mediate some of the effects of androgens.
This review focuses on mouse models showing the interactions of androgen and
growth factor pathways that promote the sexual differentiation of reproductive organs.
Recent studies investigating context dependent AR target genes are also discussed.
This article is part of a Special Issue entitled: Nuclear receptors in animal development.

All sex steroids are made intracellularly in peripheral tissues by the mechanisms of
intracrinology after menopause

Fernand Labrie
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 133–138

Following the arrest of estradiol secretion by the ovaries at menopause, all estrogens
and all androgens in postmenopausal women are made locally in peripheral target
tissues according to the physiological mechanisms of intracrinology. The locally
made sex steroids exert their action and are inactivated intracellularly without
biologically significant release of the active sex steroids in the circulation.The
level of expression of the steroid-forming and steroid-inactivating enzymes is
specific to each cell type in each tissue, thus permitting to each cell/tissue to
synthesize a small amount of androgens and/or estrogens in order to meet the
local physiological needs without affecting the other tissues of the organism.
Achieved after 500 million years of evolution, combination of the arrest of ovarian
estrogen secretion, the availability of high circulating levels of DHEA and the
expression of the peripheral sex steroid-forming enzymes have permitted the
appearance of menopause with a continuing access to intra-tissular sex steroids
for the individual cells/tissues without systemic exposure to circulating estradiol.
In fact, one essential condition of menopause is to maintain serum estradiol at
biologically inactive (subthreshold) concentrations, thus avoiding stimulation of the
endometrium and risk of endometrial cancer. Measurement of the low levels of
serum estrogens and androgens in postmenopausal women absolutely requires
the use of MS/MS-based technology in order to obtain reliable accurate, specific
and precise assays. While the activity of the series of steroidogenic enzymes can
vary, the serum levels of DHEA show large individual variations going from barely
detectable to practically normal “premenopausal” values, thus explaining the
absence of menopausal symptoms in about 25% of women. It should be added
that the intracrine system has no feedback elements to adjust the serum levels
of DHEA, thus meaning that women with low DHEA activity will not be improved
without external supplementation. Exogenous DHEA, however, follows the same
intracrine rules as described for endogenous DHEA, thus maintaining serum
estrogen levels at subthreshold or biologically inactive concentrations. Such blood
concentrations are not different from those observed in normal postmenopausal
women having high serum DHEA concentrations. Androgens, on the other hand,
are practically all made intracellularly from DHEA by the mechanisms of intracrinology
and are always maintained at very low levels in the blood in both pre- and
postmenopausal women. Proof of the importance of intracrinology is also provided,
among others, by the well-recognized benefits of aromatase inhibitors and
anti-estrogens used successfully for the treatment of breast cancer in
postmenopausal women where all estrogens are made locally. Each medical
indication for the use of DHEA, however, requires clinical trials performed
according to the FDA guidelines and the best rules of clinical medicine.
A multi-step, dynamic allosteric model of testosterone’s binding to sex hormone
binding globulin

Mikhail N. Zakharov, Shalender Bhasin, Thomas G. Travison, Ran Xue, et al.
Molecular and Cellular Endocrinology 399 (2015) 190–200

Purpose: Circulating free testosterone (FT) levels have been used widely in the
diagnosis and treatment of hypogonadism in men. Due to experimental
complexities in FT measurements, the Endocrine Society has recommended
the use of calculated FT (cFT) as an appropriate approach for estimating FT.
We show here that the prevailing model of testosterone’s binding to SHBG,
which assumes that each SHBG dimer binds two testosterone molecules
and that the two binding sites on SHBG have similar binding affinity is
erroneous and provides FT values that differ substantially from those
obtained using equilibrium dialysis.
Methods: We characterized testosterone’s binding to SHBG using
binding isotherms, ligand depletion curves, and isothermal titration
calorimetry (ITC). We derived a new model of testosterone’s binding to
SHBG from these experimental data and used this model to determine
FT concentrations and compare these values with those derived from
equilibrium dialysis.
Results: Experimental data on testosterone’s association with SHBG
generated using binding isotherms including equilibrium binding, ligand
depletion experiments, and ITC provide evidence of a multi-step dynamic
process, encompassing at least two inter-converting microstates in unliganded
SHBG, readjustment of equilibria between unliganded states upon binding
of the first ligand molecule, and allosteric interaction between two binding
sites of SHBG dimer. FT concentrations in men determined using the new
multistep dynamic model with complex allostery did not differ from those
measured using equilibrium dialysis. Systematic error in calculated FT
vales in females using Vermeulen’s model was also significantly reduced.
In European Male Aging Study, the men deemed to have low FT (<2.5th
percentile) by the new model were at increased risk of sexual symptoms
and elevated LH.
Conclusion: Testosterone’s binding to SHBG is a multi-step dynamic
process that involves complex allostery within SHBG dimer. FT values
obtained using the new model have close correspondence with those
measured using equilibrium dialysis.

Cohesin modulates transcription of estrogen-responsive genes

Jisha Antony, Tanushree Dasgupta, Jenny M. Rhodes, Miranda V. McEwan, et al.
Biochimica et Biophysica Acta 1849 (2015) 257–269

The cohesin complex has essential roles in cell division, DNA damage repair
and gene transcription. The transcriptional function of cohesin is thought to
derive from its ability to connect distant regulatory elements with gene promoters.
Genome-wide binding of cohesin in breast cancer cells frequently coincides
with estrogen receptor alpha (ERα), leading to the hypothesis that cohesin
facilitates estrogen-dependent gene transcription. We found that cohesin
modulates the expression of only a subset of genes in the ER transcription
program, either activating or repressing transcription depending on the gene
target. Estrogen-responsive genes most significantly influenced by cohesin
were enriched in pathways associated with breast cancer progression such
as PI3K and ErbB1. In MCF7 breast cancer cells, cohesin depletion enhanced
transcription of TFF1 and TFF2, and was associated with increased ER binding
and increased interaction between TFF1 and its distal enhancer situated
within TMPRSS3. In contrast, cohesin depletion reduced c-MYC mRNA and
was accompanied by reduced interaction between a distal enhancer of c-MYC
and its promoters. Our data indicates that cohesin is not a universal facilitator
of ER-induced transcription and can even restrict enhancer–promoter communication.
We propose that cohesion modulates transcription of estrogen-dependent genes
to achieve appropriate directionality and amplitude of expression.
Angiogenesis in Breast Cancer and its Correlation with Estrogen, Progesterone
Receptors and other Prognostic Factors

Jyotsna Naresh Bharti, Poonam Rani, Vinay Kamal, Prem Narayan Agarwal
Journal of Clinical and Diagnostic Research. 2015 Jan, Vol-9(1): EC05-EC07

Purpose: The  aim  of  study  is  to  evaluate  angiogenesis using  CD34,  in
estrogen,  progesterone  positive  and  negative breast cancer  and  to  correlate
the  microvessel  density  with known  histological  prognostic  factors,
morphological  type  of breast carcinoma and lymph node metastasis.
Materials and Methods: Twenty eight untreated cases of breast cancer were
included  in  the  study  and  paraffin  embedded  sections  were  obtained
from  representative  mastectomy specimen of breast cancer patient. The sections
were stained with hematoxylin and eosin stain and immunohistochemistry was
performed using CD34, estrogen, progesterone, cytokeratin and epithelial
membrane antigen  antibody.  Angiogenesis was analyzed using CD 34 antibody.
For statistical analysis, cases were grouped into estrogen, progesterone positive
and negative receptors.
Results: Mean microvessel density in ER-/PR-, ER-/ PR+, ER+/PR-, ER+/PR+
was 15.45, 14.83, 11, 10.89 respectively.  A significant correlation was found
between ER receptors and mean vascular density with p-value (< 0.05).
A significant difference was observed in mean vascular density between
the four groups comprising (p-value < 0.05).  Infiltrating duct carcinoma
(NOS) grade III has got the highest mean microvessel density (14.17)
followed by grade II (12.93) and grade I (12.33).
Conclusion: Information about prognostic factors in breast cancer
patients may lead to better ways to identify those patients at high risk
who might benefit from adjuvant therapies.

Combined blockade of testicular and locally made androgens in prostate cancer:
A highly significant medical progress based upon intracrinology

Fernand Labrie
Journal of Steroid Biochemistry & Molecular Biology 145 (2015) 144–156

Recently two drugs, namely the antiandrogen MDV-3100 and the inhibitor
of 17β-hydroxylase abiraterone have been accepted by the FDA for the
treatment of castration-resistant prostate cancer (CRPC) with or without
previous chemotherapy, with a prolongation of overall survival of 2.2–4.8months.
While medical (GnRH agonist) or surgical castration reduces the serum levels
of testosterone by about 97%, an important concentration of testosterone and
dihydrotestosterone remains in the prostate and activates the androgen receptor
(AR), thus offering an explanation for the positive data obtained in CRPC. In fact,
explanation of the response observed with MDV-3100 or enzalutamide in CRPC
is essentially a blockade of the action or formation of intraprostatic androgens.
In addition to the inhibition of the action or formation of androgens made locally
by the mechanisms of intracrinology, increased AR levels and AR mutations can
be involved, especially in very advanced disease.

Read Full Post »

Sex Hormones, Adrenal Cortisol, Prostaglandins


Curator: Larry H. Bernstein, MD, FCAP


A major class of lipids, steroids, have a ring structure of three cyclohexanes and one
cyclopentane in a fused ring system as shown below. There are a variety of functional
groups that may be attached. The main feature, as in all lipids, is the large number of
carbon-hydrogens which make steroids non-polar.

Steroids include such well known compounds as cholesterol, sex hormones, birth
control pills, cortisone, and anabolic steroids.



 The best known and most abundant steroid in the body is cholesterol. Cholesterol is
formed in brain tissue, nerve tissue, and the blood stream. It is the major compound
found in gallstones and bile salts. Cholesterol also contributes to the formation of
deposits on the inner walls of blood vessels. This topic was covered in the previous
discussion of the lipids series, and extensively in cardiovascular topics.

Cholesterol is synthesized by the liver from carbohydrates and proteins as well as fat.
Therefore, the elimination of cholesterol rich foods from the diet does not necessarily
lower blood cholesterol levels. Some studies have found that if certain unsaturated fats
and oils are substituted for saturated fats, the blood cholesterol level decreases.
The research is incomplete on this problem.

Cholesterol exists as an ester with fatty acids.What is the functional group at carbon 3
which is used to make an ester?
OH is alcohol

What is the feature on carbon 17?
Branched long hydrocarbon chain

Sex Hormones

sex hormones

sex hormones

 The primary sex hormones, testosterone and estrogen, are responsible for the
development of secondary sex characteristics. Two female sex hormones,
progesterone and estrogen or estradiol control the ovulation cycle. Notice
that the male and female hormones have only slight differences in structures,
but yet have very different physiological effects.

Testosterone promotes the normal development of male genital organs and
is synthesized from cholesterol in the testes. It also promotes secondary male
sexual characteristics such as deep voice, facial and body hair.

Estrogen, along with progesterone regulates changes occurring in the uterus
and ovaries known as the menstrual cycle. Estrogen is synthesized from
testosterone by making the first ring aromatic which results in the loss of a
methyl group and formation of an alcohol group.

List three functional groups in progesterone?
C#3 & #17 are ketones; C#4&5 are alkenes;

What is difference between progesterone and testosterone?
testosterone has C#17 alcohol vs ketone on progesterone

What is difference between testosterone and estrogen?
Estrogen has C#3 alcohol, + aromatic first ring;
no methyl group on C#17

Adrenocorticoid Hormones

The adrenocorticoid hormones are products of the adrenal glands.

The most important mineralcorticoid is aldosterone, which regulates the
reabsorption of sodium and chloride ions in the kidney tubules and increases
the loss of potassium ions.Aldosterone is secreted when blood sodium ion
levels are too low to cause the kidney to retain sodium ions. If sodium
levels are elevated, aldosterone is not secreted, so that some sodium
will be lost in the urine. Aldosterone also controls swelling in the tissues.

Cortisol, the most important glucocortinoid, has the function of increasing
glucose and glycogen concentrations in the body. These reactions are
completed in the liver by taking fatty acids from lipid storage cells and
amino acids from body proteins to make glucose and glycogen.

In addition, cortisol is elevated in the circulation with cytokine mediated
(IL1, IL1, TNFα) inflammatory reaction, called the systemic inflammatory
response syndrome. Its ketone derivative, cortisone, has the ability
to relieve inflammatory effects. Cortisone or similar synthetic derivatives
such as prednisolone are used to treat inflammatory diseases, rheumatoid
arthritis, and bronchial asthma. There are many side effects with the use
of cortisone drugs, such as bone resorption, so there use must be
monitored carefully.  Cortisol is increased pathologically with the growth
of a pituitary gland tumor that secretes adrenocorticotropic hormone
(ACTH), called Addison’s Disease, which is also associated with
hirsuit features.

What is the only difference between cortisol and aldosterone?
Aldosterone has C#13 aldehyde instead of methyl group




Prostaglandins, are like hormones in that they act as chemical messengers,
but do not move to other sites, but work right within the cells where
they are synthesized. (PARACRINE)

Prostaglandins are unsaturated carboxylic acids, consisting of of a 20 carbon
skeleton that also contains a five member ring. They are biochemically
synthesized from the fatty acid, arachidonic acid.

arachidonic acid

arachidonic acid

 The unique shape of the arachidonic acid caused by a series of cis double
 helps to put it into position to make the five member ring.

Prostaglandins are unsaturated carboxylic acids, consisting of a

  • 20 carbon skeleton that also contains
  • a five member ring and
  • are based upon the fatty acid, arachidonic acid.

There are a variety of structures one, two, or three double bonds. On the
five member ring there may also be double bonds, a ketone, or alcohol groups.

In PGE2, list all of the functional groups.
one acid; two alkenes; two alcohols; one ketone

What is difference the C=C double bonds?
the upper is cis; the lower is trans.

prostaglandin PGE2

prostaglandin PGE2

Functions of Prostaglandins 

There are a variety of physiological effects including:

  1. Activation of the inflammatory response, production of pain, and fever.
    When tissues are damaged, white blood cells flood to the site to
    try to minimize tissue destruction. Prostaglandins are produced
    as a result.
  2. Blood clots form when a blood vessel is damaged. A type of
    prostaglandin called thromboxane stimulates constriction and
    clotting of platelets. Conversely, PGI2, is produced to have the
    opposite effect on the walls of blood vessels where clots
    should not be forming.
  3. Certain prostaglandins are involved with the induction of labor
    and other reproductive processes. PGE2 causes uterine
    contractions and has been used to induce labor.
  4. Prostaglandins are involved in several other organs such as
    the gastrointestinal tract (inhibit acid synthesis and increase
    secretion of protective mucus), increase blood flow in kidneys,
    and leukotriens promote constriction of bronchi associated
    with asthma.

When you see that prostaglandins induce inflammation, pain, and fever,
what comes to mind but aspirin. Aspirin blocks an enzyme called
cyclooxygenase, COX-1 and COX-2, which is involved with the ring
closure and addition of oxygen to arachidonic acid converting to

The acetyl group on aspirin is hydrolzed and then bonded to the
alcohol group of serine as an ester. This has the effect of blocking
the channel in the enzyme and arachidonic can not enter the active
site of the enzyme.

By inhibiting or blocking this enzyme, the synthesis of prostaglandins
is blocked, which in turn relives some of the effects of pain and fever.

cox1 aspirin

cox1 aspirin


Sphingolipids are a second type of lipid found in cell membranes, particularly
nerve cells and brain tissues. They do not contain glycerol, but retain the
two alcohols with the middle position occupied by an amine.

As shown in the graphic, sphingosine has three parts, a three carbon
chain with two alcohols and amine attached and a long hydrocarbon chain.

 Structure of Sphingomyelin

In sphingomyelin, the base sphingosine has several other groups attached
as shown in the graphic on the left. A fatty acid is attached to the amine
through amide bond. Phosphate is attached through a phosphate ester bond,
and again through a phosphate ester bond to choline.

The human brain and spinal cord is made up of gray and white regions.
The white region is made of nerve axons wrapped in a white lipid coating,
the myelin sheath, which provides insulation to allow rapid conduction of
electrical signals. Multiple sclerosis caused by a gradual degradation of
the myelin sheath.

Sphingomyleins are located throughout the body in nerve cell membranes.
They make up about 25 % of the lipids in the myelin sheath that surrounds
and insulates cells of the central nervous system.

Niemann-Pick disease is caused by a deficiency of an enzyme that breaks
down excessive sphingomyelin, which then builds up on the liver, spleen,
brain, and bone marrow. An effected child usually dies within several years.



Glycolipids and Cerebrosides

Glycolipids are complex lipids that contain carbohydrates. Cerebrosides are an
example which contain the sphingosine backbone attached to a fatty acid and
a carbohydrate. The carbohydrates are most often glucose or galactose. Those
that contain several carbohydrates are called gangliosides. The example on the
left is shown with glucose. Glucocerebroside has the specific function to be in
the cell membranes of macrophages, (cells that protect the body by destroying
foreign microorganisms. Galactocerebroside is found almost exclusively in the
membranes of brain cells.

There are several genetic diseases resulting from the absence of specific enzymes
which breakdown the glycolipids. Tay-Sachs, which mainly effects Jewish children,
results in a build up of gangliosides and result in death in several years. Gaucher’s
disease results in the excessive build up of glucocerebroside resulting in severe
anemia and enlarged liver and spleen.



Read Full Post »

Larry H Bernstein, MD, FCAP, Reporter and Curator new relationship identified in preterm stress and development of autism or schizophrenia/


This is a fascinating study.  It is of considerable interest because it deals with several items that need to be addressed with respect to neurodevelopmental disruptive disorders.  It leaves open some aspects that are known, but not subject to investigation in the experiments.  Then there is also no reporting of some associations that are known at the time of deveopment of these disorders – autism spectrum, and schizophrenia.  Of course, I don’t know how it would be possible to also look at prediction of a possible relationship to later development of mood disorders.

  1. The placenta functions as an endocrine organ in the conversion of androsteinedione to testosterone during pregnancy, which is delivered to the fetus.
  2. The conversion is by a known enzymatic pathway – and there is a sex difference in the depression of testosterone in males, females not affected.
  3. There is a greater susceptibility of males to autism and schizophrenia than of females, which I as reader, had not known, but if this is true, it would lend some credence to a biological advantage to protect the females of animal species, and might raise some interest into what relationship it has to protecting multitasking for females.
  4. It is well known that the twin studies that have been carried out determined that in identical twins, there is discordance as a rule.  Those studies are old, and they did not examine whether the other identical twin might be anywhere on the autism spectrum disorder (not then termed “spectrum”.
  5. However, there is a clear effect of stress on “gene expression”, and in this case we are looking at enzymation suppression at the placental level affecting trascriptional activity in the male fetus.  The same genetic signature exists in the male genetic profile, so we are not looking at a clear somatic mutation in this study.
  6. There is also much less specific an association with the MTHFR gene mutation at either one or two loci. This would have to be looked at as a possible separate post translational somatic mutation.
  7. Whether there is another component expressed later in the function of the zinc metalloproteinase under stress in the affected subject is worth considering, but can’t be commented on with respect to the study.

Penn Team Links Placental Marker of Prenatal Stress to Neurodevelopmental Problems 

By Ilene Schneider          July 8, 2014

When a woman experiences a stressful event early in pregnancy, the risk that her child will develop autism spectrum disorders or schizophrenia increases. The way in which maternal stress is transmitted to the brain of the developing fetus, leading to these problems in neurodevelopment, is poorly understood.

New findings by University of Pennsylvania School of Veterinary Medicine scientists suggest that an enzyme found in the placenta is likely playing an important role. This enzyme, O-linked-N-acetylglucosamine transferase, or OGT, translates maternal stress into a reprogramming signal for the brain before birth. The study was supported by the National Institute of Mental Health.

“By manipulating this one gene, we were able to recapitulate many aspects of early prenatal stress,” said Tracy L. Bale, senior author on the paper and a professor in the Department of Animal Biology at Penn Vet. “OGT seems to be serving a role as the ‘canary in the coal mine,’ offering a readout of mom’s stress to change the baby’s developing brain. Bale, who also holds an appointment in the Department of Psychiatry, co-authored tha paper with postdoctoral researcher Christopher L. Howerton, for PNAS.

OGT is known to play a role in gene expression through chromatin remodeling, a process that makes some genes more or less available to be converted into proteins. In a study published last year in PNAS, Bale’s lab found that placentas from male mice pups had lower levels of OGT than those from female pups, and placentas from mothers that had been exposed to stress early in gestation had lower overall levels of OGT than placentas from the mothers’ unstressed counterparts.

“People think that the placenta only serves to promote blood flow between a mom and her baby, but that’s really not all it’s doing,” Bale said. “It’s a very dynamic endocrine tissue and it’s sex-specific, and we’ve shown that tampering with it can dramatically affect a baby’s developing brain.”

To elucidate how reduced levels of OGT might be transmitting signals through the placenta to a fetus, Bale and Howerton bred mice that partially or fully lacked OGT in the placenta. They then compared these transgenic mice to animals that had been subjected to mild stressors during early gestation, such as predator odor, unfamiliar objects or unusual noises, during the first week of their pregnancies.

The researchers performed a genome-wide search for genes that were affected by the altered levels of OGT and were also affected by exposure to early prenatal stress using a specific activational histone mark and found a broad swath of common gene expression patterns.

They chose to focus on one particular differentially regulated gene called Hsd17b3, which encodes an enzyme that converts androstenedione, a steroid hormone, to testosterone. The researchers found this gene to be particularly interesting in part because neurodevelopmental disorders such as autism and schizophrenia have strong gender biases, where they either predominantly affect males or present earlier in males.

Placentas associated with male mice pups born to stressed mothers had reduced levels of the enzyme Hsd17b3, and, as a result, had higher levels of androstenedione and lower levels of testosterone than normal mice.

“This could mean that, with early prenatal stress, males have less masculinization,” Bale said. “This is important because autism tends to be thought of as the brain in a hypermasculinized state, and schizophrenia is thought of as a hypomasculinized state. It makes sense that there is something about this process of testosterone synthesis that is being disrupted.”

Furthermore, the mice born to mothers with disrupted OGT looked like the offspring of stressed mothers in other ways. Although they were born at a normal weight, their growth slowed at weaning. Their body weight as adults was 10 to 20 percent lower than control mice.

Because of the key role that that the hypothalamus plays in controlling growth and many other critical survival functions, the Penn Vet researchers then screened the mouse genome for genes with differential expression in the hypothalamus, comparing normal mice, mice with reduced OGT and mice born to stressed mothers.

They identified several gene sets related to the structure and function of mitochrondria, the powerhouses of cells that are responsible for producing energy. And indeed, when compared by an enzymatic assay that examines mitochondria biogenesis, both the mice born to stressed mothers and mice born to mothers with reduced OGT had dramatically reduced mitochondrial function in their hypothalamus compared to normal mice. These studies were done in collaboration with Narayan Avadhani’s lab at Penn Vet. Such reduced function could explain why the growth patterns of mice appeared similar until weaning, at which point energy demands go up.

“If you have a really bad furnace you might be okay if temperatures are mild,” Bale said. “But, if it’s very cold, it can’t meet demand. It could be the same for these mice. If you’re in a litter close to your siblings and mom, you don’t need to produce a lot of heat, but once you wean you have an extra demand for producing heat. They’re just not keeping up.”

Bale points out that mitochondrial dysfunction in the brain has been reported in both schizophrenia and autism patients. In future work, Bale hopes to identify a suite of maternal plasma stress biomarkers that could signal an increased risk of neurodevelopmental disease for the baby.

“With that kind of a signature, we’d have a way to detect at-risk pregnancies and think about ways to intervene much earlier than waiting to look at the term placenta,” she said.


Read Full Post »