From the 1950s onward there has been significant headway made to define the precise role of sex chromosomes in gender disposition, improve techniques to measure and purify hormones and receptors, characterize the cellular and molecular basis of hormonal actions, and apply concepts to manipulate reproduction.
More on the Y chromosome. The Y chromosome was considered irrelevant in mammalian sex determination until 1959; that year two case reports indicated that a sterile woman was karyotype XO and a man XXY. Indirect proof of the masculinizing effect of the Y chromosome came in 1959 when Welshons and Russell demonstrated that XO mice were female; this genotype was soon verified in other mammals (in the meadow vole it is the normal female state). It is now apparent that the Y chromosome determines gonadal sex in mammals (ovaries develop in the absence of Y).
During the early 1900s units of heredity (genes) were localized to specific chromosomal regions. Many years later Watson and Crick (1953) deduced a deoxyribonucleic acid (DNA) model (based on X-ray diffraction studies of Wilkins and biochemical analyses of others) of the working structure of genes. The focus of current research is to map the genes and sequence the DNA encoding proteins that determine sex. Conserved candidate DNA sequences (mouse, human) have recently been proposed as testis-determining regions of the Y chromosome.
Radioimmunoassay. The capability to quantify small amounts of hormone in many samples was fulfilled with the advent of competitive-binding assays. The theoretical basis of radioimmunoassay (RIA) was advanced by Yalow and Berson in 1959 (they were interested in measuring plasma insulin in diabetic patients). The RIA system relies on a principle of competition between a radioactive hormone (tracer) and unlabeled hormone (standard or unknown sample) for binding sites on a specific antibody. Amount of hormone in a biological fluid is interpolated from a standard plot of tracer-bound antibody versus content of standard hormone. By 1970 RIA had been placed into large-scale practice. Early applications of RIA allowed the pulsatility of hormone secretions to be defined during conditions of health and disease. The repercussions of the RIA technique on endocrinology has been compared to the effect of Galileo's telescope on the study of astronomy.
Many variations on the RIA theme have since been devised. Although radiometric procedures are still very common, nonradiolabel assays are becoming increasingly prevalent; these techniques have advantages in that they do not generate radioactive waste and can be used outside the laboratory.
Protein hormones. With new advances in peptide chemistry, du Vigneaud in the early 1950s was able to determine the structures of two brain hormones - oxytocin and vasopressin. Finally, structural analyses of the peptide (< 30 amino acids long) and polypeptide hormones was off to a start.
Many skilled investigators labored toward the chemical purification of the gonadotropins - Choh Li was a central figure in this quest. In 1959 Li reported the structure of sheep LH. Ten years later Li described the complete primary structure of ovine prolactin. The pituitary and placental gonadotropins, but not prolactin, could be dissociated into two dissimilar subunits. Complete amino acid sequences of alpha and beta chains of the gonadotropins of several species were published by the early 1970s.
By 1960 the race was on to identify the structure of the hypothalamic factor controlling gonadotropin secretion. After better than a decade of perseverance and untold tons of hypothalamic tissues, a research team headed by Andrew Schally showed porcine GnRH to be a decapeptide (1971); the ovine structure was promptly verified by Roger Guillemin and his collaborators. Guillemin and Schally, for their research with neurohormones, and Yalow for her work on competitive-binding assays (Berson was deceased), shared the 1977 Nobel Prize for Physiology or Medicine.
Porcine relaxin was isolated and purified in 1974. Primary structures of other gonadal proteins - MIH and inhibin, were not elucidated until the 1980s.
Today purified polypeptides can be sequenced automatically; the most popular technique is the Edman degradation procedure. Amino acids are cleaved sequentially from the N-terminus of the molecule by treatment with phenylisothiocyanate and mild acid. Individual residues can then be identified by high performance liquid chromatography (HPLC).
Solid-phase methods for the chemical synthesis of peptide hormones became widely available during the 1980s. Protein hormones can now be generated using genetic engineering methods. For example, DNA encoding for a particular hormone is amplified using a procedure called polymerase chain reaction (PCR) and inserted (using restriction endonucleases) into a transfer vector (plasmid or viral DNA). Foreign protein is then expressed in vitro (Latin for "in glass") by hosts (eg., bacteria or insect cells) transfected with recombinant DNA. Recombinant technologies are rapidly finding widespread research and clinical applications.
Much is still to be learned about the secondary (folding of polypeptide chains into a helices and � pleats), tertiary (combination of secondary features into compact domains), and quaternary (organization of several chains into a single protein molecule) structures of the protein hormones - to this end modeling techniques of protein biochemistry are being perfected.
Prostaglandins. Interest in the prostaglandins was rekindled by Bergstrom and Samuelsson working at the Karolinska Institute in Stockholm during the 1960s - they revealed that these labile compounds belonged to a family of cyclic 20-carbon chain polyunsaturated fatty acid metabolites of arachidonic acid (eicosanoids; eicosa = twenty). Prostaglandin (PG) E2 and F2a were the most stable eicosanoids and the first to be synthesized in the laboratory. Thromboxanes and prostacyclin were subsequently isolated. It is now apparent that the cyclooxygenase route, which generates the above named eicosanoids, is not the only pathway of arachidonate catabolism. Arachidonic acid is metabolized by lipoxygenases to yield the hydroperoxides, hydroxy acids, leukotrienes, and lipoxins. As a group eicosanoids are produced by nearly every type of cell in the body and have been implicated in a staggering assortment of pathophysiological processes.
Within the realm of reproduction, eicosanoids are involved in ovarian functions (ovulation and luteal regression) and parturition. Though usually considered to act locally, eicosanoids can reach the circulation. Specialized utero-ovarian vascular connections allow the uterus of some species to communicate directly with the ovaries; this constitutes a mode by which uterine luteolysin (PGF2a) can access the ovaries without traversing the peripheral bloodstream.
In 1971 Sir John Vane provided evidence that the mechanism of action of aspirin and related nonsteroidal antiinflammatory drugs (NSAIDs) was due to inhibition of prostaglandin biosynthesis. Shortly thereafter, indomethacin, a potent cyclooxygenase inhibitor, was shown to block mammalian ovulation. NSAIDs have since been used to alter many reproductive processes.
Drs. Bergstrom, Samuelsson, and Vane were formally recognized for their important achievements in 1982 with the awarding of the Nobel Prize for Physiology or Medicine.
Hormonal actions. After the reproductive hormones were identified and their functions assigned, efforts were bent toward discerning the molecular steps committed to bringing about endocrine-mediated cellular responses. The process of activation begins with binding of a hormone to its matched receptor according to a "key-in-lock" analogy (a concept proposed during the early 1900s by Paul Ehrlich, an eminent immunologist) - the end-result often depends on activation (or suppression) of genomic expression.
Receptors for peptide/protein hormones and local modulators are typically membrane-associated; this was conclusively demonstrated using hormones tagged with radioisotopes. A common predated view was that hormones freely entered cells and modified metabolic processes by acting as enzyme cofactors. Attempts to purify (unstable) receptors from membranes proved difficult and unrewarding. Using innovative techniques in molecular biology, complementary (c) DNAs encoding several surface receptors, copied from corresponding messenger ribonucleic acid (mRNA) molecules, have been cloned. Deduced receptor glycoproteins are composed of an extracellular domain, a region that spans the plasma membrane, and a cytoplasmic tail.
That membrane receptors are coupled to guanine nucleotide-binding (G) proteins was recognized in the early 1970s by Martin Rodbell (a 1994 Nobel laureate). G-proteins transmit the hormonal message to enzyme systems of the cell membrane.
The field of transmembrane signal transduction was opened in the late 1950s by Earl Sutherland and his colleagues; they discovered that hormonal stimulation of a membrane enzyme (adenyl cyclase) prompted the synthesis of an intracellular "second" messenger molecule - cyclic adenosine monophosphate (cAMP) (the hormone ["first" messenger] was glucagon or epinephrine, the cells were those of the liver, and the response was glycogenolysis). The cAMP relay mechanism was later extended to many other water-soluble hormones, including those of the reproductive system.
More recently additional second-messenger systems have been distinguished, such as that involving phosphoinositide turnover. Phosphoinositide is an integral membrane phospholipid that is enzymatically processed into two second messenger molecules - inositol triphosphate (IP3) and diacylglycerol (DG). It now appears that some reproductive hormones work through the phosphoinositide system.
A third versatile second messenger is calcium. When bound to a ubiquitous intracellular protein (calmodulin), calcium plays an important role in hormone stimulus-response coupling. Intermediary effects of calcium-calmodulin in reproductive functions were first delineated during the 1970s and 80s.
How then were intracellular chemical messages, generated by membrane-active hormones, translated to protein synthesis and membrane perturbations? Initial clues came during the 1960s from work by Krebs (a 1992 Nobel laureate) and associates on cAMP-dependent protein kinases. It became apparent that second messenger molecules activate protein kinases, which phosphorylate proteins that regulate genes and membrane ion channels.
Steroid hormones have an edge on protein hormones as far as cellular accessibility. Because of their lipid solubility steroids can traverse the plasma membrane and enter cells by simple diffusion. Once inside, steroid hormones bind to specific receptors to alter the genetic programming apparatus of their target. The initial indication that steroid hormones could alter genomic expression was the finding in 1960 that the insect steroid, ecdysone, caused formation of puffs in salivary chromosomes. For years the dogma for the mechanism of steroid hormonal action was that unoccupied receptors were confined largely to the cytoplasm - the receptor was activated upon binding with hormone and then translocated into the nucleus. Later experiments indicated that detection of receptors in cytosolic fractions of cellular homogenates was due largely to contamination with material that is bound loosely within the nuclear matrix. Recent comparative structure-function analyses indicate that steroid receptors belong to a conserved family composed of three major domains - an amino terminus, a DNA-binding motif, and a carboxy terminus steroid-binding region.
Under some situations circulatory steroid hormones are converted to a more active form within a target cell; an important example is the transformation of testosterone into dihydrotestosterone (DHT) by 5a-reductase. Dihydrotestosterone is the primary nuclear androgen in male sexual tissue; it was not isolated and synthesized until 1968.
Classical (long-term) responses to steroid hormones are blocked by inhibitors of RNA (eg., actinomycin) and protein (eg., cycloheximide) synthesis. A new line of research has demonstrated that steroid hormones can alter neuronal excitability's and behaviors within seconds to minutes. Indeed, more than 60 years ago Selye reported rapid sedative-hypnotic effects of progesterone and deoxycorticosterone. Nongenomic actions of steroids appear to involve membrane receptors; additional studies will clarify the physiology of neuroactive steroid effects on these novel surface receptors.
While steroid hormones are crucial determinants in the regulation of normal reproductive processes, these same hormones can augment benign (localized tumor) and malignant (cancers that metastasize and destructively invade other tissues) diseases manifested by steroid-sensitive organs - most affected are the prostate gland, breasts, uterus, and ovaries. Steroid hormones are associated with approximately one-third of cancer cases currently diagnosed in the United States. Precisely how hormones function in the developmental mechanics and progression of cancers is intricate and related to specific type of tumor. Risk for disease is programmed to some degree by cumulative hormonal exposure. Steroid hormones can presumably act as true carcinogens by inducing mutations in DNA which result in expression of malignant phenotypes; usually however, these hormones behave as promoters in the presence of a cancer-causing agent. Steroid hormones may render growth regulatory proto-oncogenes susceptible to carcinogenic action.
Endocrine management of steroid-dependent tumors of the reproductive system has traditionally involved two approaches - surgical removal of the gonads (sometimes prophylactic in familial cases) or hormonal therapy. Pharmacological options have entailed attempts to diminish production of pituitary gonadotropins (eg., with analogs of GnRH) or directly interfere with steroid hormone action (eg., with enzymatic inhibitors of active metabolite formation or with receptor-competitive antihormones); shortcomings of these therapies can include undesirable systemic side-reactions, low efficacy, and repetitive/costly treatment regimens. The crusade continues to develop new chemopreventative agents and improve treatment outcomes related to steroid-dependent neoplasms.
Earnest attempts to quantitate receptors with radioactive hormones began in the 1950s. However, difficulties coupling hormones with radioactive atoms (diminished functional activities, nonspecific binding) were not overcome until around 1970. As early as 1949 Scatchard proposed a graphical analysis method of ligand-receptor binding that has become the standard to measure unoccupied receptors and their affinities. It is now possible to study the distribution of receptors without compromising tissue architecture; this can be done by using hormones or receptor antibodies tagged with nonradioactive compounds (eg., electron-dense molecules, such as ferritin or colloid gold).
Contraception. Numerical control of the human population has been a major medical and social concern of our time. The "baby boom" of the 1950s bore an alarm in the United States of a projected population explosion that would exceed natural resources of support. Although several means of birth control have since been achieved, safer and more effective alternatives are needed. Many third-world countries cannot adequately meet their population demand for food.
Most couples in the United States who engage in intercourse use some form of birth control (Table 1-3). The responsibility of preventing an unwanted pregnancy is one shouldered mainly by the female partner. A number of contraceptive methods are available to women - steroid hormones, the diaphragm, IUD, and vaginal spermicides. Other than the condom, no satisfactory contraceptive has been developed for use by men. Surgical sterilization, by vasectomy or oviductal ligation, has become the most popular method of pregnancy prevention among married couples in the United States; it is also the most effective means of birth control. Surgical procedures are considered permanent, though microsurgical techniques are used to realign the tubes and establish patency. Oral contraceptives are the most utilized and effective nonsurgical method of fertility regulation.
A contraceptive potential for ovarian steroid hormones was recognized early in this century by physiologists who observed that ovulation was inhibited in the presence of the CL. Chemists, such as Djerassi and Colton, synthesized orally-active compounds during the early 1950s. In 1953 Pincus and Chang reported an antiovulatory effect of oral progestins in the rabbit; this marked the beginning of intensive efforts, spearheaded by Gregory Pincus (with persuasion by Margaret Sanger, who founded the International Planned Parenthood Federation), to develop a medication for human usage. Since its introduction in 1960, "the pill," has liberated millions of women from the adversities of excessive childbearing and abortion. The untimely death of Dr. Pincus in 1967 almost assuredly cheated him of a Nobel prize.
Early versions of oral contraceptives, employing the "more-is-better" mentality, contained high amounts of steroid hormones delivered in tandem (ie., a synthetic estrogen alone followed by an estrogen-progestin combination). After a peak in popularity during the so-called "sexual revolution," oral contraceptive use subsided during the mid-to-late 1970s and into the 1980s; this coincided with a public awareness that unopposed estrogen users were at increased risk of neoplasia and cardiovascular disease. Most formulations used today are relatively low-dose estrogen/progestin combinations. The majority of recent medical surveys indicate that the infrequent detrimental effects attributed to oral contraceptives are offset by more common health benefits. Conventional regimens of three-weeks-on/one-week-off (eg., Ortho Tri-Cyclen, Yasmin) are designed to mimic a 28-day menstrual cycle. Seasonale, a 12-weeks-on/one-week-off oral contraceptive system (translating to four menstrual periods per year), received Food and Drug Administration (FDA) sanction in 2003. Seasonique, an update version of Seasonale, employs a very low estrogen dose during the week of menses - which may reduce symptoms of hormonal withdrawal (cramping and migraine) and stabilize the uterine lining (prolonged progestin exposure can cause atrophy and break-through bleeding). Shorter/lighter periods are manifested by the 24/4-day dosing schedules of Yaz and Loestrin 24 Fe approved in 2006 and 26/2 regimen of Natazia approved in 2010. Lybrel, a continuous daily low-dose estrogen/progestin pill (there are no inactive/placebo pills in the packets) designed to halt menstrual periods indefinitely, was approved in 2007. Progestin-only "mini-pills," which are taken continuously, are an option for (eg., lactating) women whom should avoid estrogens.
Several long-acting (female) steroidal preparations also are available. Depo-Provera, an injectable form of medroxyprogesterone acetate (MPA) administered every three months, was approved (after more than two decades of testing) by the FDA in 1992. Implanon (now called Nexplanon), a three-year progestin-releasing subcutaneous implant, was approved in 2006. An estrogen/progestin-releasing intravaginal ring (NuvaRing, which is left in place for three weeks) and a transdermal patch (OrthoEvra, which is applied once a week for three out of four weeks) were approved in 2001.
Two-treatment prescription regimens of steroidal combination (Preven Kit) and progestin-only (Plan B) oral contraceptive pills were approved in 1998-99 by the FDA for "emergency contraception." The concept is to delay or prevent ovulation (or interfere with fertilization) soon (within three days) after unprotected intercourse. Two doses were given 12 hours apart. After a contentious effort to ease access to an emergency contraceptive, Plan B was approved in 2006 for behind-the-counter sales. A one-pill version (1.5 mg levonorgestrel) of Plan B was approved in 2009; it is available without prescription for women 17 years old or older. A new type of prescription emergency contraceptive, containing an antiprogesterone - ulipristal acetate (to be sold under the brand name Ella), was approved in 2010. Ella can prevent pregnancy up to five days after intercourse and is slightly more effective than Plan B. It appears that the primary mechanism of action is to counteract implantation.
The successes of steroidal contraception in women have not been replicated in men. The predicament is that the male produces massive numbers of gametes. Furthermore, it can require almost a month for spermatogenesis to be halted, and once it has, sperm cells can persist within the epididymides and vas deferens for several additional weeks (this explains Aristotle's confusion regarding an obligatory role of the testis in fertility). Therefore, chronically high blood levels of estrogen-androgen combinations (to effectively inhibit gonadotropin secretion) must be attained to induce azoospermia. Unacceptable side-effects of steroid hormones include impotence and feminization.
Intrauterine devices, while very effective and once popular, have all but disappeared as an optional method of contraception in the United States. For fear of lawsuits concerning complications, and poor corporate profits, most firms have ceased manufacturing IUDs. Some initial hysteria stemmed from the use of the Dalkon shield during the early 1970s (Figure 1-3). Only two prescription devices are currently obtainable on the US market - Mirena (a five-year progestin-releasing system) and ParaGard (12-year copper T380A). Intrauterine devices continue to be an important form of contraception in many developing countries, such as China.
Other mechanical methods of contraception, most notably the condom, while less effective than the IUD, have gained in use; a contributing factor to this trend has been the AIDS epidemic. The condom is the only contraceptive with the added advantage of significant protection against sexually-transmissible diseases. Latex rubber condoms were introduced after the innovation of vulcanized rubber during the 1940s.
Several spermicides have been tested over the years; these agents are applied to the vagina before coitus. Spermicides are merchandised as foams, creams, jellies, and suppositories; they are most effective when used with a barrier contraceptive. A nonprescription form of spermicide-containing sponge is also available in the United States. The active ingredient in most modern-day spermicides is nonoxynol-9 or octoxynol. A few early studies linked the use of spermicides at conception with congenital (dating from birth) malformations; more contemporary, better-controlled investigations have not corroborated this relationship.
The rhythm (fertility awareness) method stands as the only contraceptive approach sanctioned by the Roman Catholic Church. The original calendar method relies on calculation of a fertile period during which the couple should abstain from intercourse. Over the last two decades adjunct natural family planning techniques have been developed to reduce the inherent error rate of forecasting ovulation; these methods include monitoring consistency of cervical mucus and basal body temperature. Cervical mucus becomes copious and slippery before ovulation. Basal body temperature increases (~ 0.6� F) with formation of the CL (a hypothalamic effect mediated by progesterone); this marks the end of the fertile period. Effective use of symptothermal indices necessitates training and motivation.
Many contraceptive techniques are in the experimental stage. There has been some work in engineering microvalves and biodegradable implants that could be placed within the vas deferens or oviducts. In the male there are prospective uses for injectable synthetic progestin-androgen combinations, GnRH analogs (combined with androgen replacement), inhibin, and gossypol. Gossypol is a pigment of cottonseed that inhibits spermatogenesis without altering circulatory testosterone; much of the research with this compound has been carried out in China. The initial excitement over gossypol has been tempered by occasional symptomatic hypokalemia (severe blood potassium depletion manifested by episodes of muscular weakness, tetany, and postural hypotension) and irreversible infertility. Antimicrobial spermicides, that protect against sexually-transmitted diseases, are being tested. An exciting new venture is the formulation of antifertility vaccines to gamete antigens, hormones, and receptors.
(Additional information on human contraceptives can be found on the Planned Parenthood [http://www.plannedparenthood.org/index.htm] website.)
Fertility control in pets, farm animals, and wildlife are also important societal concerns. In the United States nearly 30 million dogs and cats are impounded annually, of which almost 20 million are euthanized (total costs incurred ~ $500,000,000). Stray animals create a health hazard by urinating and defecating in public places and inflict millions of bites (some lethal) on people each year. Additionally, millions of dollars are lost yearly to farmers and ranchers as a result of livestock predation by feral dog packs and coyotes. The mainstay approach to control, surgical sterilization, is labor-intensive and not without risk to the animal. There is pain and suffering that accompany castration or ovariohysterectomy; incision dehiscence, infection, and even death, are not uncommon. Surgical castration (to reduce aggressive behaviors and undesirable carcass traits) or spaying (to prevent estrus and feedlot pregnancy) of farm animals is routinely performed by unqualified personnel under less than desirable conditions. Many consider castration by vascular emasculation with rubber bands to be cruel. A diversity of methods have been applied to control surplus populations of free-roaming wildlife; lethal techniques include shooting, trapping, and poisoning (eg., with sodium cyanide or fluoroacetate) - terrestrial or aerial shooting is often of limited impact and impractical, trapping can be inhumane and its lingering exploit is being challenged, and use of toxicants has met with resistance because of environmental repercussions. Evolving techniques in animal biotechnology should furnish us with more humane nonsurgical contraceptives that can be efficiently and safely implemented.
Induced abortion. About two-thirds of births involving unmarried women in the United States are unwanted. One-in-ten teenage women become pregnant each year. Over one-third of pregnancies among married women are unplanned. Obviously sex education and contraception have fallen short of completely protecting women from unwanted pregnancies - hence therapeutic abortion.
Is it a woman's prerogative to decide the fate of her unborn, or is this decision one of governmental jurisdiction? World-wide laws controlling abortion range from permissive to very restrictive. About one-half of the world's women live in countries where abortion is available upon request; another one-fourth live in areas where abortion is only permitted to protect a woman's health. The most prohibitive policies are found in fundamentalist Islamic nations and countries of Latin America and sub-Saharan Africa. An estimated 50 million abortions are carried out each year - nearly half are illegal.
Elective abortion, with restrictions, was legalized in the United States in 1973 (Supreme Court cases Roe [a pregnant single women who brought a class action challenging the constitutionality of Texas criminal abortion laws] vs. Wade [District Attorney of Dallas County] and Doe vs. Bolton). Abortion for unwanted pregnancy was allowed during the first trimester of gestation; procedures performed after this time are permitted only in cases of physical or mental endangerment to the woman or if a fetal defect is detected. Federal moneys for abortion were strictly limited by Congress in 1976 (Hyde Amendment). Some states continue to fund abortions for impoverished women. In 1989 the Supreme Court (Webster vs. Reproductive Services) sustained a Missouri law forbidding the execution of abortions by public employees or in taxpayer-funded facilities. In 1992 the Court upheld the 1973 rulings, but granted further state controls. Individual states can set specific requirements regarding residence, parental/spousal notification, and a (24-hour) waiting period. More than one million legal abortions are performed in America every year; this represents almost one abortion per four live births.
The conventional method to terminate a first trimester pregnancy has been by D & C. The D & C approach is being replaced by less traumatic procedures; metal dilators can damage a stenotic cervix, rendering it incompetent and unable to support future pregnancies (premature birth). Many clinicians now use a prostaglandin-releasing vaginal suppository to help dilate the cervix. Alternatively, a Laminaria tent (a cylinder of dried, sterilized seaweed that absorbs water and expands) or inflatable rubber catheter can be inserted into the cervix before surgical or suction curettage. Vacuum aspiration is more efficient with fewer complications than traditional sharp curettage. Mortality rates due to legal abortion performed during the first trimester are lower (< 2/100,000 woman/year) than those associated with child birth (~ 5-22/100,000 woman/year, risk increasing with age).
Uterine muscle stimulants, such as prostaglandins, oxytocin, and estrogenics are abortifacient; these compounds are being tested as postcoital interceptive ("morning-after" pill) or contragestational ("once-a-month" pill) agents. The antiprogesterone RU 486 (mifepristone), in combination with an orally-active prostaglandin (misoprostol), has been used extensively (since 1988) in France (where it was developed at the pharmaceutical firm Roussel-Uclaf), the United Kingdom, Sweden, and China; its heralded licensing in the United States (conditional approval was granted by the FDA in 1996 and finalized in 2000) has met with opposition - though it may be useful in the treatment of some cancers (another anticancer drug, methotrexate, which interferes with uterine and embryonic growth, is a possible option to RU 486). Clinical trials are underway using a vaccine against the b subunit of hCG (again, resentment toward its abortive action may restrict availability in this country).
Dangers of abortion increase with duration of pregnancy. Until recently, infusion of hypertonic saline solution into the amniotic cavity was used to induce precocious labor and delivery during the second trimester. Most second trimester abortions are now induced with prostaglandins. Partial-birth abortions, usually performed during the fifth or sixth month of gestation, involve extricating the brain of the fetus within the cervical canal before completing a vaginal delivery. A limited number of third trimester abortions are carried out in the United States; when done, these are usually accomplished by hysterotomy (removal of the fetus through an abdominal incision).
Very few social issues are as volatile as that concerning elective termination of pregnancy and its legality. There continues to be a stalwart right-to-life movement in this country to make abortion prohibitive. Opponents of legalized abortion contend that it involves the intentional killing of a human being. The prochoice stance is that abortion is a basic right of privacy. Complex ethical and religious emotions center on this controversy, which I will not debate. Two points are clear: 1) abortions will not end with legislation; and 2) the life of a woman is put into jeopardy when confronted with an illicit abortion. An absolute ban on abortion could outlaw any contraceptive measure that can act after fertilization (eg., ovarian steroids and IUDs).
In 1988 the Reagan-Bush administration banned federal funding of research using fetal tissues obtained by induced abortions. The Clinton-Gore administration lifted this moratorium with stipulations - an informed consent to terminate a pregnancy must be kept independent from the (uncompensated) prospective use of tissue (transplantation of fetal tissues could be of value in the treatment of heart and kidney failure, Parkinsonism, Alzheimer's disease, diabetes mellitus, and leukemia). During his first week in office, President George W. Bush signed an executive order banning federal support for international family-planning organizations that perform abortions or offer abortion counseling.
Assisted reproduction. Infertility among the livestock species and within the human population is a recurring enigma. An aim of assisted reproduction is to overcome infertility's that plague animals and man.
Efficiency of reproduction is a vital factor that determines the economic viability of any animal production enterprise. Despite advances, major barriers limiting maximal productivity persist (Table 1-4); these complex physiological problems will be resolved only after sound fundamental knowledge is accrued through basic research. New techniques in molecular biology will afford livestock producers the opportunity to select breeding stock on genotype rather than phenotype.
More than 15% of all married couples in the United States who wish to have children experience some form of infertility (ie., the inability to conceive after one year of unprotected intercourse). A myriad of etiologies having an anatomical, congenital, endocrinological, metabolic, psychological, immunological, and(or) inflammatory basis can lead to failure of pregnancy (Table 1-5). Overall, about 70% of cases of human infertility can now be treated with success.
Progestin supplementation (feeding, injection, subcutaneous implant, intravaginal device) and withdrawal has been used experimentally to regulate the onset of estrus in farm animals for almost 50 years. More recently, combinations of steroid hormones, gonadotropins, and PGF2a have been used to control estrous cycles. Prostaglandin (Upjohn) received FDA approval for use in beef cattle and nonlactating dairy heifers in 1979. The steroidal Syncro-Mate-B system (Ceva; Sanofi; Rhone Merieux) has since been added to (and recently removed from) the repertoire of commercially-available cattle synchronization agents. The Eazi-Breed CIDR (Pharmacia) intravaginal progesterone-releasing device was approved in 2002 for estrous synchronization in beef cattle and dairy heifers.
An assortment of hormonal treatments have been devised to induce ovulation in anovulatory women. Customary tactics are to stimulate the ovaries directly with exogenous gonadotropins (pergonal) or to enhance endogenous production of gonadotropins with GnRH, inhibitors of negative estrogen feedback (clomiphene, tamoxifen), or an inhibitor of prolactin secretion (bromocryptine). A risk of ovarian hyperstimulation is superovulation and multiple births. Concepts regarding endocrine therapy of the infertile male have developed since the mid-1970s. Unfortunately, management of male hypogonadism with hormones has not been especially effective.
Artificial insemination was one of the earliest methods used to manipulate reproduction. In farm animals AI is the most powerful tool available to make rapid genetic progress and it is an effective means to control venereal diseases. Nevertheless, outside the (intensive) dairy industry, commercial use of AI is still quite limited (eg., < 5% of beef females are inseminated artificially each year). In humans intrauterine insemination of concentrated sperm cells can be used in cases of oligospermia (low sperm count) or when cells are unable to permeate the cervical mucus.
Sexing of sperm cells has been a long-time goal of the AI industry. Preselection of gender has significant application in farm animals - choice of males or females for meat or milk production, respectively. Several techniques have been designed to prepare enriched populations of cells bearing either an X or Y chromosome. Most methodologies have relied on the fact that spermatozoa carrying the Y chromosome are slightly lighter (ie., have less DNA) and more motile than X cells. Significant shifts in sex ratios have been reported for laboratory animals, but progress with farm species has been painstakingly slow. Cells can be sorted by flow cytometry or immunoaffinity chromatography using antibodies directed against cell-surface antigens encoded specifically by a sex chromosome. Cells also can be removed from embryos by biopsy to determine sex (and screen for genetic defects) - an intervention first reported using human embryos in 1989.
The shortcoming of AI alone is that it does not take advantage of the equally important contribution to the genetic makeup of the offspring made by the female. Although the first embryo transfer was reported over a century ago, the technology heeded little attention before 1950. Techniques have been developed to collect ova or embryos from outstanding donors for transfer to (genetically-inferior) recipients. The first calf produced from a frozen embryo was reported in 1973. Because transfer procedures require considerable skill and are expensive, only specialized applications in research and seedstock breeding have been realized.
In vitro fertilization (IVF) and embryo transfer (ET) procedures, once the exclusive domain of animal husbandry, have been mainstreamed into human clinical medicine. First-line approaches to establish patency in blocked oviducts of women has been by gas infusion, transcervical balloon tuboplasty, or surgical reconstruction - if the prognosis was in doubt (eg., extensive bilateral intraluminal obliteration) then IVF-ET became a prospect. In the IVF process the father's sperm is used to fertilize the mother's ova (collected by follicular aspiration) in vitro. The embryo is then deposited into the uterus, bypassing the occluded oviducts. On July 25, 1978, the world's first "test-tube" baby, Louise Joy Brown, was born in Bristol, England; this remarkable feat, orchestrated by Steptoe and Edwards, was the end-result of research that began in 1944 when Rock and Menkin reported the fertilization of a human egg in vitro. Millions of IVF babies have been born worldwide. The first baby born from a frozen embryo was reported in Australia in 1984. The rate of success of IVF-ET is ever increasing with refined technique - about 40% using fresh embryos. In vitro fertilization is now implemented empirically under circumstances of unexplained infertility. Donor sperm and ova can be used in cases of gonadal resistance to hormonal treatments. Surrogate mothers have carried IVF babies to term for their biological parents. The cost per pregnancy via IVF-ET is approximately $50,000 (ovulation induction + intrauterine insemination = $10,000-15,000).
Modifications of the IVF-ET procedure have come into practice. Gamete intrafallopian transfer (GIFT), introduced in 1984, is an alternative when a significant portion of at least one oviduct is open to the uterine cavity; the technique is similar to that of IVF-ET (and cheaper), except that washed spermatozoa and ova are placed in the oviduct at laparoscopy. Thus, with GIFT, fertilization takes place in vivo ("in the living body"). With zygote intrafallopian transfer (ZIFT) ova are fertilized in vitro and immediately placed into the oviduct below the blockage. Microinjection of a spermatozoon directly into the ooplasm (intracytoplasmic sperm injection, ICSI) can be used when donor spermatozoa are otherwise incapable of penetrating the ovum (the first child was born to ICSI in 1992). In vitro maturation (IVM) is a technique that avoids complications of ovarian hyperstimulation; ova of immature follicles are collected and cultured in a maturation-inducing medium (supplemented with gonadotropin and/or growth factors) before ICSI/IVF-ET.
Even more elaborate techniques of preimplantation embryonic micromanipulation have been devised. Identical offspring (clones) can be generated by cutting embryos and transferring the embryonic cells into enucleated eggs. Embryos of mice were first cloned in 1970. Sheep and cattle embryos were cloned in 1979 and 1980. Human blastomeres were separated and cloned in 1993. A rather astonishing innovation to the cloning/embryo transfer repertoire was reported in 1997 - the birth of a lamb derived from the DNA code of an adult mammary cell electrofused to an enucleated ovum … asexual reproduction in a mammal (Figure 1-4)!
Researchers are able to inject genes into fertilized ova which become integrated into the genome of the embryo by random recombination of homologous DNA sequences; although inefficient (usually < 5%), this can yield founder transgenic (recombinant) animals that pass this new genetic material to the next generation by sexual reproduction. Targeted gene transfer has also been accomplished using modified retroviruses (vectors that contain reverse transcriptase, and therefore can make DNA using their RNA as a template), nuclear transplantation from pluripotent embryonal stem cell lines containing a foreign DNA insert, or by electroporation into fertilizing sperm cells. Efforts are underway to increase the efficiency of transfection by site-specific gene constructs. A recent report indicates that transformation efficiency can be improved markedly when a gene is simply introduced into a periovulatory (unfertilized/metaphase-arrested) ovum (in which the nuclear membrane is disintegrated - and thus, easier for the foreign DNA to access the chromosomes).
Mice were the first transgenics - produced in the early 1980s. By the mid-1980s transgenic farm animals (containing extra copies of a metallothionein-human growth hormone fusion gene) were born. The hope is that transgenic technology will allow for the agricultural production of animals that grow and reproduce more efficiently and are resistant to disease and stress. Perhaps valuable pharmaceuticals can be over-expressed by transgenes in "bioreactor" animals (eg., within milk). Transgenic pigs are being developed as sources of organs for human transplantations.
Emerging revolutionary methods in reproduction have raised some serious ethical, theological, and legal questions that have not yet been adequately addressed. Should transgenic animals be patented? Will reproductive technologies have negative effects on the environment or the quality of our food supply? Should infertile couples be bestowed the liberty to bear children by artificial means - or is this immoral meddling? Will in vitro technologies lead to selective breeding in humans? Should innovations be made available only to those who can afford it? What about custody rights of donors and surrogates? Should restrictions be enacted if individuals are motivated purely by monetary gain? How will embryos not transplanted be handled and disposed of? What are the consequences of asexual cloning? Is the destiny of human reproduction (as Aldous Huxley prophesied in his book Brave New World) to spawn in "hatcheries?"
Persistent political pressure has been applied by some activist groups to halt all experimentation dealing with assisted reproduction - both in vivo and in vitro. The platform (since 1979) is that research on external fertilization be sanctioned to counteract infertility, but that human embryos fertilized in the laboratory not be maintained beyond two weeks (ie., the approximate time implantation is completed in vivo). Initially, the National Institutes of Health allowed federal funds to be used for research with stem cells obtained from surplus (frozen) human embryos (undifferentiated cells extracted from embryos, which destroys the embryo in the process, raising moral and ethical concerns, can be prompted in culture to grow into many different tissues - the goal is to use this technology as a source of replacement cells to treat diseases). This stance was then limited by the second President Bush to preexisting cell lines. President Obama lifted the federal restrictions on stem cell policies imposed by the Bush administration. President Clinton placed a ban on human cloning research which was later sanctioned by the National Bioethics Advisory Committee.