The Current State of Hormonal and Nonhormonal Male Contraception

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THE CURRENT STATE OF HORMONAL AND NONHORMONAL MALE CONTRACEPTION BY CATHERINE BUCHANAN MCGRATH A THESIS Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Elzie McCord, Jr. PhD Sarasota, Florida April, 2010

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ii Dedication T h i s w o r k i s d e d i c a t e d t o t h e H o o t e n a n n y H o u s e a n d a l l o f t h e w o n d e r f u l e x p e r i e n c e s i t s i n h a b i t a n t s h a v e g i v e n m e

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iii Acknowledgements A s p e c i a l t h a n k s t o m y c o m m i t t e e m e m b e r s D r A m y C l o r e D r S a n d r a G i l c h r i s t a n d e s p e c i a l l y D r E l z i e M c C o r d J r w i t h o u t w h o m I m a y h a v e h a d a c o m p l e t e l y d i f f e r e n t a n d p o t e n t i a l l y l e s s f u l f i l l i n g N e w C o l l e g e e x p e r i e n c e T h a n k y o u t o a l l m y f r i e n d s f a m i l y a n d a d o p t e d f a m i l i e s f o r t h e i r l i m i t l e s s e m o t i o n a l s u p p o r t A n d f i n a l l y I w o u l d l i k e t o t h a n k J e f f B o i s s o n e a u l t f o r h i s u n f a i l i n g l o v e

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iv Table of Contents DEDICATION ................................ ................................ ................................ ................................ ..... II ACKNOW LEDGEMENTS ................................ ................................ ................................ ................... III ABSTRACT ................................ ................................ ................................ ................................ ........ IX INTRODUCTION ................................ ................................ ................................ ................................ 1 BACKGROUND ................................ ................................ ................................ ................................ .. 3 BARRIER METHODS ................................ ................................ ................................ .......................... 8 STERILIZATION ................................ ................................ ................................ ............................... 18 WITHDRAWAL ................................ ................................ ................................ ................................ 28 FERTILITY AWARENESS BASED METHODS ................................ ................................ ..................... 31 MALE HORMONAL CONTRA CEPTIVES ................................ ................................ ........................... 37 MALE NONHORMONAL CON TRACEPTIVES: IMMUNOC ONTRAC EPTION ................................ ...... 47 MALE NONHORMONAL CON TRACEPTIVES: PLANT D ERIVED COMPOUNDS AND INDAZOLE CARBOXYLIC ACIDS ................................ ................................ ................................ ......................... 57 CONCLUSIONS ................................ ................................ ................................ ................................ 62 REFERENCES ................................ ................................ ................................ ................................ ... 65

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v List of Tables Table 1: Perfect and Typical Use Efficacy Rates of Male Condoms ................................ ............... 10 Table 2: Perfect Use Efficacy of Female Barrier Methods ................................ ............................. 17 Table 3: Efficacy Rates of Male Sterilization Techniques ................................ ............................... 20 Table 4: Perfect and Typical Use Efficacy Rates for Condoms compared to Withdrawal ............. 30 Table 5: Perfect and Typical Use Efficacy Rates for FABMs ................................ ........................... 33 Table 6: Percentage of Asian and Caucasian men reaching oligozoospermia and azoospermia within 4 mo. of weekly TE injections ................................ ................................ ............... 39

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vi List of Figures Figure 1: Structural organization of the human seminiferous tubule ................................ ............. 3 Figure 2(a d): Insertion instruct ions for female barrier methods ................................ ................. 15 Figure 3: The vas deferens is immobilized under the skin with a clamp ................................ ....... 18 Figure 4: Layers surrounding the vas deferens ................................ ................................ .............. 21 Figure 5(a f): Vasal disruption methods for vasectomy. ................................ ............................... 22 Figure 6: Beaded bracelet used in SDM to keep track of the f ertile period ................................ .. 32 Figure 7: Hormonal, follicular, temperature, secretions, and cervical changes during the menstr ual cycle. ................................ ................................ ................................ ............. 34 Figure 8: Sperm oocyte interaction ................................ ................................ ............................... 48 Figure 9: Molecular structure of Gossypol. ................................ ................................ ................... 58 Figure 10: Molecular structure of tripdiolide ................................ ................................ ................ 59 Figure 11: Molecular structure of triptolide ................................ ................................ .................. 59 Figure 12: Molecula r structure of adjudin ................................ ................................ ..................... 61

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vii List of Abbreviations ASA Anti Sperm Antibody BBT Basal Body Temperature Method BTB Blood Testis Barrier CatSper Cation Channel of Sperm DMPA Depot Medroxyprogesterone Acetate ENG Et onogestrel Eppin Epididymal Protease Inhibitor ES Ectoplasmic Specializations FA 1 Fertilization Antigen 1 FABM Fertility Awareness Based Method FI Fascial Interposition FSH Follicle Stimulating Hormone GnRH Gonadotropin Releasing Hormone hCG Human Chorionic Gonadotropin HDL C High Density Lipoprotein Cholesterol ISO International Organization for Standardization IUD Intra Uterine Device IVD Intra Vas Device LDH Lactate Dehydrogenase LDH C 4 Lactate Dehydrogenase C 4 LE Ligatio n/Excision LH Luteinizing Hormone LHRH Luteinizing Hormone Releasing Hormone

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viii LNG Levonorgestrel MENT Methyl 19 nortestosterone MHC Male Hormonal Contraception NSFG National Survey of Family Growth PSA Prostatic Specific Antigen RISUG Reversible Inhibition of Sperm Under Guidance SDM Standard Days Method SEBS Styrene Ethylene Butylene Styr ene Sg Semenogelin STI Sexually Transmitted Infection T Gel Testosterone Gel TDM Two Day Method TE Testosterone Enanthate TGF Transforming Growth Factor TU Testosterone Undecanoate US FDA United States Food and Drug Administration VLP Virus like Particle YLP YLP 12 ZP Zona Pellucida

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ix THE CURRENT STATE OF HORMONAL AND NONHORMONAL MALE CONTRACEPTION Catherine Buchanan McGrath New College of Florida, 2010 A BSTRACT The unintended pregnancy (UIP) rate in the United States is ne arly 50%. To reduce UIP, a diverse range of contrac ep tive options should be available to men and women. Studies have shown that the availability of a diverse range of contraceptive options for both men and women increase s the rate of correct and consiste nt usage, and may contribute to a decrease in the unintended UIP rate. The female contraceptive market is vast and includes the female condom, hysterectomy, and steroids delivered via pills, patches, vaginal rings, implants, injections, and Intra Uterine Devices. Male directed methods are comparatively less diverse and are limited to two forms: the male condom and vasectomy. Although several traditional methods exist where male participation is necessary (e.g. withdrawal, fertility awarenes s based methods), the male contracep tive market suffers from a lack of reversible non coital dependent opt ions. The addition of hormonal (i.e. Androgen only, Androgen Progestin, and GNRH regimens) and non hormonal methods (i.e. immunocontraception, plant derived compounds, and indazole ca rboxylic acids ) should significantly address the unmet need for men seeking alternative contraceptive methods.

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x This review investigates and presents the current contraceptive options available to men and o utlines those under development. ___________________ _______ Dr. Elzie McCord, Jr. Division of Natural Sciences

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1 CHAPTER 1 I NTRODUCTION Forty nine percent (49%) of all pregnancies in the United States are unintended and half of those end in abortion (Fin er and Henshaw 2006). While abortion is a safe procedure, it can cost $350 $900 and carry a social stigma (Planned Parent Hood 2009). The availability of effective male and female contraceptives has the potential to further family planning efforts and redu ce the unintended pregnancy rate. No single contraceptive method is appropriate for every man or woman; therefore, a variety of contraceptives that are safe, eff ective, and congruous with user lifestyles should be made available. Indeed, the ability to c ustomize contraceptive methods decreases the likelihood of medical complications and increases the likelihood of correct and consistent regiment compliance (Trussell 2004). Current contraceptive methods can be loosely categorized into barrier, surgical, and nonsurgical methods. The female contraceptive market is vast and includes the female condom, hysterectomy, and steroids delivered via pills, patches, vaginal rings, implants, injectables, and Intra Uterine Devices (IUDs). Male directed contraceptives are comparatively much less diverse and come in only two forms: the male condom and vasectomy ( Trussel and Wynn 2008 ; Naz and Rowan 2009 ). The male condom has been in use for centuries (Youssef 1993). It is a well known, reliable, and easy to use form of birth control with particularly high use in developed countries (Anderson and Baird 2002). Latex condoms are 86% effective in preventing pregnancy with typical use and 98% effective with perfect use (Trussel and Wynn 2008 ; Naz and Rowan 2009 ). Whereas c ondoms prov ide temporary contraception at the time of coitus, vasectomies are associated with a one time procedure that results in permanent contraception. Vasectomies involve the division, occlusion, or cautery of the vas deferens (Anderson and Baird

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2 200 2) through an invasive procedure that is not easily reversed. Vasectomies can have efficacy rates as high as 99.5%; however, rates vary according to the surgeon and technique used and can be as low as 61% (Sokal and others 2004). Reversal surgery is possi ble but has a low success rate; therefore, vasectomy targets men who have achieved their ideal family size (Naz and Rowan 2009). Couples seeking additional or alternative contraception to the condom or vasectomy often choose female contraceptives due to their availability. It is estimated that 44% of couples using contraceptives in developed countries rely on female contraceptives compared to 26% on male contraceptives. Partners willing to share contraceptive responsibility would benefit from the introd uction of additional effective male contraceptive methods. A series of contraceptives that are diverse and widely available are better suited to meeting the needs of men at various stages of sexual activity and should reach a wider audience. A high prior ity has been placed on the development of new and alternative forms of male contraception (Anderson and others 2002).

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3 CHAPTER 2 BACKGROUND The average male produces daily about 100 200 million spermatozoa, mature germ cells cr itical to reproduction (Mr uk 2008) Spermatozoa begin as undifferentiated germ cells in the testicular seminiferous epithelium (Fig. 1A) and mature within approximately 64 days in the Seminifer ous Epithelial Cycle Spermatogenesis and spermiogenesis are two components of the Semi niferous Epithelial Cycle. Spermatocyte to spermatid maturation requires 24 days and spermatid to spermatozoa maturation requires 5 weeks in spermatogenesis and spermiogenesis, respectively (Mruk 2008). Figure 1 : (A) Structural organization of the human seminiferous tubule. (B) S tages of s perm maturation during the Seminiferous Epithelial Cycle (Alberts and others 2002)

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4 The Seminiferous Epithelial Cycle Spermatogenesis is the process by which immature germ cells, known as sper matogonia, proliferate and differentiate into primary and secondary spermatocytes, spermatids, and spermatozoa as they migrate from the base of the seminiferous tubule toward the lumen (Nussey and Whitehead 2001; Johnson and others 2007). Mitotic prolifer ation of spermatogonia begins in the epithelial lining of the seminiferous tubule s of the testes when males near puberty. The spermatogonia undergo meiotic division to produce primary and secondary spermatocytes, which become spermatids (Fig. 1 B ) (Nussey and Whitehead 2001). The gametes move away from the lining and toward the lumen of the tubules with each stage of maturity. The primary spermatocytes must overcome the blood testis barrier (BTB), an immunological barrier similar in function to the blood brain barrier, as they transition toward the lumen. The BTB is composed of Sertoli cells, tight junctions, adherens junctions, desmosome like junctions, and gap junctions, which undergo substantial restructuring to allow for primary spermatocyte passage i nto the adluminal region (Alberts and others 2002; Cheng and others 2009). Spermatogonia maturation is largely dependent on the pituitary gonadotropin follicle stimulating hormone (FSH). In the absence of FSH, spermatogenesis occurs at a severely depre ssed rate. Another pituitary gonadotropin, luteinizing hormone (LH), along with intratesticular testosterone are critical in the later stages of spermatogenesis, particularly spermiogenesis (Matthiesson and others 2006). Spermiogenesis is the final stage of spermatogenesis. Spermatids mature at this stage into spermatozoa via elongation of the sperm tail condensation of nuclear material in the head and the formation of the acrosome (Sofikitis and others 2008; Mruk and others 2008). Spermatozoa formation initiates spermiation

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5 in which the germ cells detach from the seminiferous epithelium and pass into the epididymis for further maturation and storage (Alberts and others 2002; Cheng and others 2009). Spermatogenesis and spermiogenesis are structurally an d metabolically support ed by Sertoli cells ( Alberts and others 2002 ; Mruk 2008 ). Sertoli cells (Fig. 1 A ) are located in the seminiferous tubules and surround the developing gametes. They stimulate a variety of processes critical to spermatogenesis, includ ing the development of primordial germ cells into spermatozoa and the production of Leydig cells. Leydig cells are located between the seminiferous tubules (Fig. 1A) and secrete the androgen testosterone, which is responsible for the development of struct ures in the male reproductive tract including the prostate and seminal vesicles. Testosterone also assists in the masculinization of the developing male brain; i.e. sexual identity, orientation, and behavior (Alberts and others 2002). Intratesticular testo sterone exists in concentrations 50 100 times higher than those found in the blood and is critical for spermatid elongation and adhesion to Sertoli Cells (Cheng and others 2009). Low levels of testosterone prevent germ cells from completing spermiogenesis and can result in apoptosis (cell death) or phagocytosis in the lumen of the seminiferous tubules (Sofikitis and others 2008). The Seminiferous Epithelial Cycle is composed of several morphological phases that are easily distinguishable, although most of the exact biochemical and molecular mechanisms involved are still relatively unknown. Of the processes mentioned, many potential targets exist that can be manipulated to produce infertility in men. The following male contraceptive review illustrates sev eral identified targets and the compounds or techniques used to induce infertility in mice rats, and men Contraceptive Efficacy Contraceptive efficacy is a measure of how well a particular method prevents pregnancy.

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6 Contraceptive efficacy rates are ac quired from surveys conducted by the National Survey of Family Growth (NSFG) and from clinical trials. The NSFG is conducted every ~7 years (the latest statistics should be available in May 2010) and includes questions that illuminate trends in birth rate s, including contraception, sexual activity, and infertility (CDC/National Center for Health Statistics 2009). Clinical trials involve human testing of experimental drugs or treatments in four phases. Phase I trials evaluate safe dosages, side effects, a nd safety in groups of 20 80 people. Phase II trials evaluate efficacy and safety in groups of 100 300. Phase III trials compare the experimental drug or treatment to pre existing ones in large groups of 1,000 3,000 people. Phase IV trials are post mark eting studies which evaluate the optimal drug or treatments, risks, and benefits (Clinical Trials 2007). Many experimental male contraceptives are undergoing Phase III trials. The risk of pregnancy with each treatment is determined via a variety of measu res such as contraceptive failure rates during typical use and during perfect use. Typical use is characterized by incorrect or inconsistent contraceptive usage. Perfect use is characterized by following the package directions. Typical use estimates ind icate the contraceptive effectiveness when used incorrectly/inconsistently. Perfect use estimates indicate the efficacy of the device (Trussell 2009). The distinction between effectiveness and efficacy can be vague and is not always identified by researc hers. Finally contraceptive efficacy can be determined by pregnancy rate or by sperm count. The pregnancy rate is a common method for efficacy determination; however, those rates are often confounded by the use of additional methods such as withdrawal or condoms (Trussell 2009). Sperm counts taken at regular intervals following initiation of treatment provide an accurate representation of the likelihood of pregnancy. Most non barrier male contraception aim to achieve azoospermia where there is no sperm found in the ejaculate. Oligospermia (<1

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7 million sperm/mL semen) may also be an acceptable level of sperm suppression. Oligospermia is associated with efficacy rates of 97 100% (Liu and others 2008).

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8 CHAPTER 3 BARRIER METHODS Introduction Barrier con traceptive methods prevent the passage of spermatozoa into the vagina by means of a physical barrier. A variety of devices exist for use in women; however, the condom is the only device available to men and it is by far the most popular of all barrier met hods. Condoms exist in either male or female forms and are the only form of contraception (excluding abstinence and a mutually monogamous sexual relationship) that can prevent the spread of most sexually transmitted infections (STIs); including those tra nsmitted via secretions (HIV, gonorrhea, etc.) and those transmitted via skin/mucosal membrane contact (Herpes simplex virus, human papilloma virus) (Mindel and Sawleshwarkar 2008; Horner and others 2009). Male Condoms The male condom is composed of a t hin layer of latex or nonlatex material that encases the glans and shaft of the penis (Gallo and others 2003). The first reported use of a male condom was in 1564 in which Gabriello Fallopio, an Italian anatomist, alleged that a moistened sheath of linen placed over the glans penis during coitus provided prot ection against venereal disease. Therefore, the initial uses of the condom were to prevent disease transmission rather than pregnancy ( Dalsimer and others 1973). In addition to linen, early condoms w ere made from oiled silk paper, tortoise shell, horn, fine leather, or animal intestines (Youssef 1993). Natural membrane, or "skin" condoms, can be made from the caecum (intestinal membrane) of lambs, calves, and goats, as well as from fish bladders (Yo ussef 1993). Their origins are unknown, although references of a sheath made from goats' bladder were made

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9 circa 150 A.D. in the Legend of Minos, King of Crete, as told by An toninus Libralis ( Dalsimer and others 1973 ; Youssef 1993 ). I. Latex Condoms Lat ex condoms constitute the majority of the commercially available condoms in the U.S. They have the highest efficacy rates of all barrier methods. Latex condoms are popular with both men and women and they have been mass produced since the mid 1800's (Gal lo and others 2003). The development of the latex condom in the 1850's was made possible by the advent of rubber vulcanization in which the properties of rubber are stabilized by heating with sulfur and other chemicals ( Dalsimer and others 1973). Althoug h initially crude in design, improvements in production efficiency and quality (shape, shelf life, and tensile strength) allowed for mass production (lower prices) and increased popularity (Youssef 1993). Today, latex condom production is fully automated and consists of molds dipped in liquid latex ( Dalsimer and others 1973; Youssef 1993). Latex condom efficacy testing is a rigorous process. Male latex condoms that are used cor rectly and consistently have a 98 % efficacy rate ( Table 1 ) Efficacy rates de crease to approximately 8 5% with typical use (Naz and Rowan 2009). Any decrease in condom effectiveness today stems mostly from inconsistent usage L atex breakage and slippage from the penis during coitus each oc cur 2% of the time (Warner 2007). Early latex condoms were made by grinding up, dissolving, and heating natural rubber with solvents, then dipping cylindrical molds into the solution and submitting them to v ulcanization (Dumm and others 1974). Unfortunat ely, these condoms deteriorated rapidly and lacked the tensile strength needed to be reliable. Liquid natural rubber latex was introduced in the 1930's and has increased in tensile strength. Manufacturing conditions were not well controlled during this

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10 t ime and dust particles would frequently contaminate the latex solutions, leading to small pin holes in the final product. Estimates in the 1930's stated that only ~40% of manufactured condoms were "fit for use" ( Dumm and others 1974). Today, condoms are quality controlled according to an international standard developed by the International Organization for Standardization (ISO). The United States uses the ASTM standard, which is very similar to ISO (Gerofi and Wong 2009) Table 1 : Perfect and Typical Use Efficacy Rates of Male Condoms (Naz and Rowan 2009; Gallo and others 2008; Walsh and others 2003; Frezieres and others 1999) Efficacy Rates Condom Material % Perfect Use % Typical Use Latex 98 99.3 85 92.1 Polyurethane 96 95 .2 Styrene Ethylene Butylene Styrene (SEBS) 94.9 98 89.2 Condom testing is conducted by both manufacturers and governments Testing protocols attempt to mimic the stresses encountered during everyday use and include tests for holes, the inflation test and a package seal test (Gerofi and Wong 2009). Government testing is usually done via sampling methods in which a small number of condoms are randomly chosen from a batch and tested for pin holes and bursting strength (Dumm and others 1974). Bursting strength is determined via the inflation test in which the condom is filled with air at ~0.5L/sec until breakage occurs (Gerofi and Wong 2009). The sampled batch of condoms may or may not be accepted depending on the existence of or the number of defectiv e condoms found. Manufacturer testing is usually electronic and includes both sampling tests and screening tests (Smith and others 1999) P in hole tests are conducted on small lots of condoms filling the condom with 300 mL of water and rolling it on ab sorbent paper to visually check for leaks. Although the risk associated with leaks is not as high as that associated with breakage, efforts to keep the number of holes below the ISO's upper limit of 0.25% are substantial M any

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11 manufacturers maintain a pi nhole rate of 0.08% (Gerofi and Wong 2009). Such sampling tests entail intensive manipulation and handling of each condom, making them unsuitable for use Screening tests on much larger batches of condoms require limited handling Condoms are placed ov er a metal mandrel and immersed in a water bath, where the current flow between the inside and outside of the condom is measured in a conductivity test (Smith and others 1999) Some countries conduct aging tests with condoms to determine the rate of deter ioration in hot (>70 C) and aerobic environments ( Dumm and others 1974). Companies such as ENERSOL, founded by Dr. John Gerofi, Australia's National Representative on the ISO's Condom Standards Committee, can be hired by manufacturers and international aid agencies to provide quality testing services. ENERSOL provides on site condom testing according to current ISO standards, consulting services that include condom plant design and manufacture, maintenance and calibration services for condom testing equ ipment, and training programs for manufacturer employees (ENERSOL 2009). Male latex condoms have a series of disadvantages that may contribute to inconsistent usage or increased failure rates. Seventy five percent ( 75% ) of men in the U.S. claim decreased sensitivity during coitus while wearing latex condoms. Many also c laim decreased sexual enjoyment. L atex is known to conduct heat poorly and requires tight fitting in order to retain its position on the penis during coitus. Similarly, at maximum stretc h, latex condoms have decreased strength and are prone to breakage (Gallo and others 2003). Increased breakage rates have also been linked to condom storage conditions. Latex has low stress tolerance. E xposure to excessive heat or aerobic environments r esults in a stiffening of the material due to oxidation (Free and others 1996). Finally, 1 3% of Americans have a latex allergy, a statistic that increases to 6 7% with greater exposure to latex (i.e. hospital workers) (Gallo and others 2003).

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12 There is a need for condoms with superior qualities. II. Nonlatex Condoms Nonlatex condoms, including natural membrane, polyurethane film, and synthetic elastomer condoms, attempt to surpass the limitations of latex condoms, particularly via increased sensatio n, hypoallergenic materials, and decreased susceptibility to deterioration Natural membrane condoms were widely used prior to the production of latex condoms in mid 19th century and are still used today T hey account for 5% of commercially produced cond oms in the U.S. and are currently only made from lamb caeca. Natural membrane condoms generally cost twice as much as latex condoms because they require individual treating and testing in processes that are labor intensive. Natural membrane condoms c anno t be mass produced as easily or effectively as latex condoms. The production of skin condoms involves rinsing the caecum with clean water immediately following the death of the lamb, fat trimming, salting, mucus and fat removal, cutting to the appropriat e length, testing with compressed air, and packaging. Users contend they transmit body heat more effectively than latex and are less constrictive on the glans penis. There are even claims that they stimulate the vaginal mucosa, although they are so far s cientifically unfounded ( Dumm and others 1974). Studies investigating the efficacy of natural membrane condoms as a barrier to semen are rare; however, several studies have indicated that they may be ineffective at preventing the transmission of STIs and HIV ( Conant and others 1984; Minuk and others 1987; Lytle and others 1990). The lamb caecum condoms are variably porous and manufacturers cannot guarantee consistent protection (Lytle and others 1990). In addition to natural membrane condoms, two types of nonlatex condoms are sold in the United States: polyurethane and a synthetic elastomer known as styrene ethylene butylene

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13 styrene (SEBS) or "Tactylon ". Both the polyurethane and the SEBS condoms are non allergenic and low profile (odorless, thinner me mbrane, transparent), and unlike latex condoms, they can be used with oil based lubricants (Walsh a nd others 2003). Polyurethane and SEBS condoms are known to conduct heat better than latex, be less constricting, and have an increased shelf life (Gallo an d others 2003). Polyurethane condoms account for <2% of condom s sold in the U.S. and offer a 95 96% efficacy rate ( Table 1 ) In general, polyurethane condoms are associated with a higher breakage rate during inte rcourse when compared with latex condoms. Interestingly, Gallo and others (2008) found no difference in typical use efficacy rate s between latex and polyurethane condoms. Co ndoms made from SEBS offer a 9 5 98 % perfect use efficacy rate ( Table 1 ), but they are associated with a low rate of vaginal irritation and itching (Walsh and others 2003). Female Barrier Methods A much wider variety of female barrier methods are available to women, yet none have achieve d the wide scale popularity of male condoms. As of 2002, less than 3% of American women use cervical barrier methods as their main form of contraception (Narrigan 2006). Although female barrier methods represent a small user population, they play a key role in preventing pregnancy and STI transmission, particularly for women who are unable to negotiate the use of alternative contraceptive methods (Padian and others 2007). An exhaustive review of female barrier methods is beyond the scope of this paper; however, several methods are worth ment ioning for comparative purposes. They includ e female condoms, diaphragms, cervical caps, and sponges. Table 2 lists a comparison of perfect use efficacy rates for female barrier con traceptives.

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14 I. Female Condoms Female condoms were approved by the U nited S tates F ood and D rug A dministration (US FDA) in 1993 for use as a barrier contraceptive to prevent STIs (Valappil and others 2005). They consist of a polyurethane or synthetic lat ex, called nitrile, sac with two soft rings that are inserted into the vagina (Fig. 2 a ). Both the polyurethane and nitrile models have performed similarly in efficacy trials ( Table 2 ) with ~97 % efficacy rate (Bek sinska and others 2005). Similarly to male condoms, female condoms are readily available and can be purchased in a drugstore or acquired from the local health department. The female condom's popularity has not reached that of male condoms, probably due i n part to the need for insertion instruction s and practice in order to reach the stated efficacy rates. However Beksinska and others (2008) found that male partners can and do assist in the proper insertion of the device (Valappil and others 2005; McNeil 2007). Unlike male condoms, high female condom failure rates are confined to inexperienced couples. Valappil and others (2005) found that first time users of the female condom who required multiple attempts at correct insertion were more likely to expe r ience increased slippage rates indicating that user skills may be important in achieving documented efficacy rates of the female condom.

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15 Figure 2 (a d): Insertion instructions for female barr ier methods: female condom (a), diaphragm (b), cervical cap (c), and contraceptive sponge (d) (Zieve and others 2008 ; Mayo Clinic staff 2010)

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16 II. Diaphragms The diaphragm is a latex or silicone device (often used in conjunction with a spermicide jelly ) which covers the cervix, creating a seal and thereby preventing semen from entering the upper reproductive tract (Fig. 2 b ) (Smith and others 1995; Padian and others 2007 ). It can be covertly worn for up to 24 hours without leading to toxic shock syndrom e and has the advantage of providing protection against HIV and STI transmission. Diaphragms must be obtained from health professionals because they require a clinical examination for fi tting and insertion instruction ; however, they are reusable for up to 3 years. Diaphragms offer an 80 96% efficacy rate ( Table 2 ) (Narrigan 2006; Black and Kubba 2008). III. Cervical Caps Two cervical caps are available in the United States: Lea's Shield and FemCap Both wer e developed by physicians a nd are similar to the diaphragm with several key differences. The cervical cap is shaped like an elliptical bowl with a mold ed ring for easy removal (Fig. 2 c ). I t contains a reservoir for spermicide and a central valve to relie ve pressure and facilitate cervical secretion drainage into the vagina (Narrigan 2006). Cervical caps range in effectiveness from 77 96% (Narrigan 2006; Black and Kubba 2008).

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17 Table 2 : Perfect Use Efficacy of Female Barrier Metho ds (Narrigan 2006; Black and Kubba 2008; Beksinska and others 2005; Trussell 2009; Trussell and others 1993) Efficacy Rates Barrier Method % Perfect Use % Typical Use Female Condom 97 79 Diaphragm 80 96 84 92 Cervical Cap 90 96 77 88 Contraceptive Sp onge 80 91 68 88 IV. Sponges The contraceptive sponge is a single use polyurethane foam cushion containing one gram of the spermicide nonoxynol 9, which is p laced against the cervix (Fig. 2 d ). The sponge is ~2.5 x 5.5cm and acts to chemically, and to a lesser extent physically, block passage of the sperm (Kuyoh and others 2003; Narrigan 2006). Contraceptive sponges are most effective in nulliparous women those who have not given birth. Sponges are 84% effective in nulliparous women and 68% effective in parous women (Narrigan 2006). Conclusions Multiple forms of male and female barrier contraceptives are available in the United States which represent a key role in low cost family planning as well as STI and HIV prevention. Female barri ers vary in de sign and efficacy but fill s a need for low profile contraceptives that women can initiate and control, particularly when hormonal methods are not an option. M ale barrier methods vary little but are highly effective when used correctly and consistently. They are widely recognized to be cheap, ubiquitous, and efficacious. In addition, they hav e the added benefit of prov iding visible proof of efficacy. T hey do n o t require medical examination, supervision, or follow up for use, and they a re small and disp osable (Youssef 1993).

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18 CHAPTER 4 STERILIZATION Introduction Male sterilization, or vasectomy, is the division and/or the obstruction of the vas deferens (Fig. 3 ) I t is utilized by 7% of couples worldwide, and is currently the only approved form of permanent contraception available to men (Anderson and Baird 2002). In the United States, over 500,000 vasectomies are performed each year (Eisenberg and others 2009). The majority of men receiving vasectomies are older than 35, married, and have had more than two children. Vasectomies are particularly common among white American males (14.1%), but less so in black (3.7%) and Hispanic (4.5%) males. Vasectomies have also been shown to be more Figure 3 : The vas deferens is immobilized under the skin with a clamp. An incision or p uncture is made in the scrotal wall and the vas is teased through (Johns Hopkins Medicine 2010)

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19 prevalent among higher income males, regardless of race (E isenberg and others 2009). A vasectomy is traditionally performed under local anesthesia. A surgical incision in the scrotum is made to access the vas deferens for occlusion A new no scalpel approach utilizes pointed forceps, decreasing bleeding and in fection risks normally associated with incisions (Anderson and Baird 2002; Labrecque and others 2004). A number of vasectomy techniques are practiced in the United States, including ligation, excision, cautery, facial interposition, and open ended vasecto my (Sokal and Labrecque 2009). Vasectomies are typically quoted as having one of the highest contraceptive efficacy ratings ( 99.9%) (Trussell and Wynn 2008). H owever, the manner in which unsuccessful procedures are reported, the technique(s) used to perf orm the surgery, and the skill of the surgeon may contribute to substantially reduced efficacy rates (Trussell and Wynn 2008) I. Ligation and Excision Ligation and excision techniques are used by ~14% of surgeons in the United St ates (Sokal and Labrecq ue 2009). It is the most common method used worldwide (Cook and others 2009) The vas deferens are ligated with either two metal clips or with suture materials and the segment between the two ligatures (0.5 4.0 cm in length) is often excised (Fig. 5 e ) ( Sokal and Labrecque 2009). Ligation and excision techniques are associated with the highest rate of failure among vasectomy methods (up to 38% Table 3 ). Failures are due, i n part to the ligation technique (Baro ne and others 2003; Sokal and others 2004; Sokal and Labrecque 2009). Excessive suture or clip pressure on the vas deferens can lead to ischemia (localized poor blood circulation), causing necrosis of the tissue and increasing the likelihood of the two c ut ends reforming. Too little pressure on the vas may allow spermatozoa to pass and thus may be considered an unsuccessful vasectomy (Sokal and Labrecque 2009). The length of the excised

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20 vas deferens segment plays a role in procedural failure. V as defer ens segment s longer than 4.0 cm are typically associated with increased efficacy rate, although there is an increased rate of surgical complications (Sokal and Labrecque 2009). Table 3 : Efficacy Rates of Male Sterilization Techniq ues (Sokal and others 2004; Song and others 2005; Sokal and Labrecque 2009; Labrecque and Caron 2004; Barone and others 2004) Technique Efficacy Rate (%) Ligation/Excision (LE) 62 87.3 LE + Fascial Interposition (FI) 85 94.1 Cautery 90 95.6 Cautery + F I 98 99.5 Intra Vas Device 73.6 II. Cautery Cautery of the vas lumen is practiced by 71% of surgeons in the United States, often in conjunction with clips or ligation, and has a n efficacy rate of 9 0 9 5.6 % ( Table 3 ) (Sokal and others 2004; Barone and others 2004; Sokal and Labrecque 2009). Tr aditionally, the vas deferens are cut and the ends are cauterized either thermally with a hot wire or electrically (Fig. 5a and 5 d ) (Sokal and Labrecque 2009). Cauterizatio n without cutting the vas is possible but is associated with a n 15% occlusive failure rate (Labrecque and Caron 2004). Cautery of the vas deferens results in a higher rate of occlusive efficacy when compared with ligation and excision with or without effi cacy enhancing techniques such as fascial interposition described below (Sokal and Labrecque 2009). Cautery techniques have also been shown to reduce the time needed to reach oligo and azoospermia. Eighty five percent (85%) of cauterized men achieved a zoosp ermia within 12 weeks compared to only 60 70 % with excision/ligation Infertility is achieved in a similar time frame as cauterized men (Barone and others 2004). In addition, fewer complications (such as spermatic granuloma, chronic inflammatory mas ses) are associated with thermal cautery when compared with elect rical (Caldwell and others 1996; Sokal and Labrecque

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21 2009 ). III. Fascial Interposition Fascial interposition is a method of increasing vasectomy effectiveness by placing a portion of the vas sheath, called the internal spermatic fascia (Fig. 4 ) between the two cut ends of the vas deferens, providing an additional barrier against sperm (Sokal and others 2004; Sokal and Labrecque 2009). E ither the testicular end or the prostatic end of th e vas deferens is placed under the spermatic fascia (Fig. 5 c ) It is sealed via a clip or with suture material. Fascial interposition has been shown to increase the effectiveness of ligation and excision (Fig. 5 a) (~85% effective), as well as ca utery (Fi g. 5 b) (99.5% effective) ( Table 3 ) (Labrecque and others 2004; Sokal and others 2004; Sokal and Labrecque 2009). A similar technique involves folding back one or both of the vas segments to provide an additional b arrier. Efficacy rates of this technique are varied and inconclusive, although they may depend on the skill of the surgeon (Trussell and Wynn 2008) Figure 4 : Layers surrounding the vas deferens (Benderev 2008)

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22 Figure 5 (a f) : Vasal disruption methods for vasectomy (Dassow and Bennett 2006).

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23 IV. Experimental Methods Several newer methods that have a high potential for reversibility involve the placement of materials into the vas deferens to block passage (Anderson and Baird 2002; Aitken and others 2008). These materials are sometimes called intra IVD consists of two silicone plugs inserted into th e vas lumen wh ich allow the passage of fluids while trapping sperm, potentially reducing the likelihood of epididymitis (inflammation of the epididymis) and other short term complications associated with the previously mentioned techniques (Song and others 2006; Rabe 2007; Aitken and others 2008). Reversible inhibition of sperm under guidance (RISUG) involves an injection of styrene maleic anhydride in a dimethyl sulfoxide vehicle directly into the vas deferens (Sohini and others 2009) This method destro ys the plasma and acrosomal membranes of sperm as they pass through the material and does not lead to the formation of Anti Sperm Antibodies (ASAs). The exact mechanism is unknown but the observed effect could be due to the copolymer's low pH that may aff ect sperm function. Anhydride is also hydrolyzed in water into the positively charged hydride which could interfere with the negatively charged sperm membranes leading to cell lysis. This method of contraception can be reversed by flushing the vas with a solution of sodium bicarbonate to dissolve the polymer ( Rabe 2007; Mr uk 2008; Aitken and others 2008 ). RISUG is currently undergoing phase III clinical trials in India (Sohini and others 2009). Male sterilization has a lower cost, minimal complications and higher success rates than female sterilization, yet the latter accounts for more than 66% of the sterilization procedures performed worldwide (Melville and Bigrigg 2008) In an effort to improve the acceptability of vasectomy, completely non invasiv e techniques involving ultrasound or lasers are being tested which eliminate the risk of infection, scrotal pain, and bleeding (Cilip and others 2009) Both methods heat the vas deferens to produce thermal coagulation, necrosis, and scarring, resulting

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24 in occlusion. Side effects of these techniques include insufficient occlusion and scrotal skin burns (Cilip and others 2009). The Efficacy of Vasectomy The efficacy of vasectomy is often determined by the skill and experience of the surgeon. Many failure rates can be partially attributed to surgical errors including but not limited to, conducting vasectomy twice on the same vas deferens, conducting vasectomy on a structure that is not the vas deferens, and overlooking a congenital duplication of the vas deferens (Labrecque and others 2006). Many of the vasectomy techniques are not standardized and the variability may contribute to the range of efficacy rates for each technique (Sokal and others 2004). Researchers most often conduct 1 2 post vasectomy se men analyses in which azoospermia or low counts of non motile sperm (less than 100,000 sperm/mL) are determined. Vasectomy success is ultimately determined by the absence of pregnancy. Post vasectomy pregnancy rates are commonly quoted as less than 1% (T russell and Wynn 2008) H owever, most of the published data are based on individual physician accounts (Labrecque and others 2006) Semen analysis techniques and follow up measures, if used, vary widely and are often poorly recorded. Few physicians util ize frequent semen analyses. They instead judge failure rates by the number of unplanned pregnancies, the r esults of which are subject to the frequency of intercourse, the female partner's fertility level, etc. This method to determine vasectomy success is largely d ependent on voluntary reports Unstable marriages may encourage female partners to have an abortion without informing her partner. Also, pregnancies occurring one year or more after a vasectomy may not be reported to the same clinic in which the vasectomy was performed (Labrecque and others 2006) At home sperm k its are currently available. They will allow men to conduct their own

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25 semen analyses to ensure azoospermia (Labrecque and others 2006). However, it is generally recommended to wai t 10 12 weeks or 15 20 ejaculations before relying on vasectomy as the primary form of birth control (Barone and others 2004). Complications Occlusion of the vas deferens is typically more reliable than ligation and excision as the two ends of the vas ca n reform and allow passage of sperm (Anderson and Baird 2002). The reforming of the vas deferens' distal and proximal cut ends is called recanalization. Recanalization can occur when various tissues and cells such as spermatozoa, epithelial cells, and co nnective tissue participate in a granulatomous reaction, bridging the gap between the two vas deferens ends (Sokal and Labrecque 2009). Epithelial microtubules proliferate through the granuloma and produce a fistula allowing the passage of sperm (Labrecqu e and others 2006). Up to 25% of ligation and excision vasectomies are complicated by recanalization in less than 12 weeks after the procedure (Sokal and Labrecque 2009) Late recanalization, occurring 12 weeks or more after vasectomy is rare and occurs in 1 in 2000 men ( Labrecque and others 2006; S okal and Labrecque 2009 ). This risk decreases when fascial interposition is used and becomes lowest when thermal cautery is used in conjunction with fascial interposition. Identification of many causes of vas ectomy failure, including recanalization, is often limited to histopathological studies of specimens collected during reversal surgeries or repeat vasectomies. Many clinicians and researchers use their own criteria to determine whether or not recanalizati on has occurred. Recanalization may be indicated by the absence or rare occurrence of sperm followed by an increase in sperm after the first two weeks post vasectomy, or simply the absence of motile sperm followed by their reappearance. Clinicians have a lso classified recanalization as a sperm counts of 1 million/mL or less followed by sperm counts of 10 million/mL or more (Labrecque

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26 and others 2006). Side Effects Vasectomy side effects can vary for each individual, but are dependent (like efficacy) on the skill of the surgeon and the type of procedure used (Anderson and Baird 2002). Vasectomy complication rates range from 1 6% (Melville and Bigrigg 2008) Sterilization complications may a lso include short term bleeding hematoma, and sperm granuloma. Longer lasting side effects are epididymitis and chronic testicular pain (Sokal and Labrecque 2009). Short term postvasectomy pain is common in ~30% of men (Tandon and Sabanegh 2008) Long term postvasectomy pain occurs in ~1 in 1000 men. Many theori es exist as to the cause of postvasectomy pain, some of which are controversial. Following vasectomy, testicular histology studies have shown dilation of the seminiferous tubules, which may or may not cause the associated interstitial fibrosis leading to pain. Also, the nerves around the vasal transection can become encased in fibrous tissue and are subject to distortion, angulation, and lymphatic infiltration, which could also be a source for pain (Tandon and Sabanegh 2 008). An increase in epididymal pr essure due to the closed end may also lead to chronic pain (Melville and Bigrigg 2008) A technique called open ended vasectomy (Fig. 5 f) in which the testicular end of the vas deferens is left unobstructed is associated with decreased epididymal damage a nd chronic pain. This method also requires less surgical time, results in a higher percentage of successful reversal surgery and does not lead to higher failure rates when the prostatic end is closed via cautery and fascial interposition (Sokal and Labre cque 2009). A caveat of vasectomy is that it disrupts the blood testes barrier by allowing sperm to exit the reproductive epithelium. Sperm are highly antigenic and initiate an inflammatory response when they leak out of the testicular end of the vas de ferens (Tandon and Sabanegh

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27 2008). Many men (60 80%) produce antisperm antibodies in response to this sperm exposure and continue to do so even after reversal surgery (Anderson and Baird 2002; Tandon and Sabanegh 2008). Consequently, the return to suffic ient fertility levels is often challenging. Some studies have debated the association of vasectomy with an increased prevalence of prostate and testicular cancers (Anderson and Baird 2002). Limited vasectomy efficacy data are caused by incomplete follow up. Many couples that experience pregnancy post vasectomy do not return to the same clinic. Patient studies detailing specific techniques (many physicians use a combination of techniques) are lacking as well as detailed long term follow ups (Sokal and La brecque 2009). Conclusions Vasectomy can be up to 99.5% effective and provides the individual with permanent contraception that does not require c ompliance at the time of coitus compared with condoms. Unfortunately, due to the low success rate of revers al surgery, vasectomy is not an ideal contraceptive for those wishing to postpone child bearing. It is also expensive and provides no protection against STIs. However, the popularity of male sterilization in both developed and developing countries sugges ts that many men are willing to participate in family planning and thus would benefit from additional methods of male contraceptives (Anderson and Baird 2002).

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28 CHAPTER 5 WITHDRAWAL Introduction Coitus Interruptus, also known as withdrawal, has been emp loyed since biblical times and is one of the most widely used contraceptive methods (Mosher and oth ers 2004), in developed and developing countries (Horner and others 2009). The concept is simple: the male partner withdraws prior to ejaculation during unp rotected vaginal intercourse. If the male partner consistently withdraws at the appropriate time, efficacy rates rise as high as 96 % (Hatcher and others 2007). Judging the moment of imminent ejaculation is often difficult, especially for first time users First year method efficacy rates are as low as 73 % (Woods and others 2009), although subsequent typical use rates average at ~82 % (Jones and others 2009). These figures suggest that some degree of prowess is necessary to prevent the passage of seminal fluid into the vagina. The Sexual Response During copulation, the male undergoes four phases of sexual response: sexual arousal, plateau, orgasm, and resolution (Zuckerman and others 2003). Upon sexual stimulation, the Cowper's gland produces a clear mu coid fluid, known as pre ejaculate, that functions to prepare the urethra for the passage of sperm via lubrication and neutralizing the acidity from residual urine (Zuckerman and others 2003; Chudnovsky and Niederberger 2007). Stray sperm in the pre ejacu late may contribute to increased failure rates; however, debate over whether sperm has been found in the pre ejaculate still exist. Masters and Johnson (1966) have traditionally claimed that the penis can sometimes discharge pre ejaculate containing low l evels of sperm;

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29 however, studies within the last 20 years have shown an absence of sperm in pre ejaculate of fertile men (Pudney and others 1992; Ilaria and others 1992 ; Zuckerman and others 2003 ). Residual sperm from a previous ejaculation may remain in the urethra if not "flushed out" by urination; therefore, if intercourse is initiated during this time, the sperm may be transported into the vagina within pre ejaculate (Zuckerman and others 2003). Efficacy Limited research exists on the efficacy of withdrawal. Many including both users and researchers do not consider withdrawal a form of contraception (Doherty and Stuart 2009). In addition, there is some degree of ambiguity attached to the term 'withdrawal'. Withdrawal can refer to one of two meth ods; the first involves removing the penis to ejaculate outside of the vagina The second involves removing the penis to apply a condom, then ejaculating within the vagina Coitus interruptus is often used in combination with coitus dependent methods, su ch as the female birth control pill and fertility awareness based methods (Schearer 1978; Jones and others 2009 ). Withdrawal has been shown to be used as a temporary contraceptive when condoms are unavailable or as a long term solution. The use of withdr awal as a contraceptive may even compete with condom usage and lead to inconsistent condom use, particularly in young adults (Horner and others 2009). The higher estimates of withdrawal efficacy (96% and 82% for perfect and typical uses, respectively) are comparable to perfect and typical use rates of latex condoms ( Table 4 ). Other estimates for withdrawal efficacy are as low as 73%, giving them one of the highest failure rates of both male and female contraceptiv es, similar to the spermicides and fertility awareness based methods (Trussell 2009). Studies often fail to clarify the term 'withdrawal' and thus efficacy and use rates range substantially and may be unreliable (Jones and others 2009).

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30 In addition to t he protection against conception, some researchers suggest that withdrawal offers some protection against both male to female (Richters 1994 ; Donovan 1995 ) and male to male (Jin and others 2010) transmission of HIV, provided there is no exposure to lesions ulcers, or semen. Despite these data, it is important to note that increased withdrawal use is correlated with an increased risk of contracting an STI (Sznitman and others 2009). Table 4 : Perfect and Typical Use Efficacy Rates f or Condoms compared to Withdrawal (Trussell 2009; Jones and others 2009) Efficacy Rates Contraceptive Method % Perfect Use % Typical Use Male Latex Condom 98 99.3 85 92.1 Withdrawal 96 73 82

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31 CHAPTER 6 FERTILITY AWARENESS BASED METHODS Introduction Fertility awareness based methods (FABMs) are unconventional contraceptive methods that rely on vigilant observation of the physical symptoms which accompany ovulation. Identification of these symptoms can be used to determine when the woman is most fer tile ( Grimes and others 2005 ; Frank Herrmann and others 2007; Pallone and Bergus 2009 ). The chance of pregnancy is reduced when additional forms of contraception, such as condoms or abstinence are employed during the woman's most fertile periods. Althoug h this method is not male directed, it requires male participation in order to be effective. Fertile periods are characterized by the presence of the ovum, which can survive up to 24 hours following ovulation. Sperm can survive up to five days in the rep roductive tract (Wilcox and others 1995 ; Germano and Jennings 2006 ) There are six days per month where sex should be avoided to prevent pregnancy. FABMs use various techniques to determine when in the woman's monthly cycle the 6 days occur (Pallone and B ergus 2009). I. Calendar Method The oldest FABM is called the calendar, or rhythm, method and was developed by Ogino and Knaus in the late 1920's (Ogino 1928; Knaus 1929 ). The method involves keeping careful records of the woman's menstrual cycle for 6 12 months. If the cycle is consistent in length, the female may predict the time of highest fertility and use backup contraception or abstinence during this time. Studies evaluating the calendar method often fail to use a set of standardized rules for de termining the period of highest fertility; however, they usually involve counting the

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32 number of days in the cycle, starting with the day of menstruation (Pallone and Bergus 2009). The Standard Days Method (SDM) is a modern form of the calendar method and employs the us e of a beaded bracelet (Fig. 6 ) to keep track of the days following menses. It was developed by Georgetown University's Institute of Reproductive Health in response to the need for standardization. A simple FABM was required that could be included in national and institutional family planning policies. The FABM is intended for women whose menstrual cycles last 26 32 days and involves an abstinence period on days 8 19 of the cycle. The first year perfect u se failure rate for SDM ( Table 5 ) is ~5% (Arevalo and others 2002; Gribble and others 2008 ). Figure 6 : Beaded bracelet used in SDM to keep track of the fertile period (Arevalo and others 2002) The efficacy of the calen dar method relies on the regularity of the menstrual cycle, which for many women, can be variable in duration. Factors such as child birth, breast feeding, menopause, the use of hormonal contraceptives, stress, and illness can lead to cycle irregularity. This method is therefore less reliable than the following FABMs (Pallone and Bergus 2009).

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33 Table 5 : Perfect and Typical Use Efficacy Rates for FABMs (Pallone and Bergus 2009; USAID and others 2007; Arevalo and others 2002; Gribbl e and others 2008) Efficacy Rates Contraceptive Method % Perfect Use % Typical Use FABMs 75 Calendar (SDM) 95 Basal Body Temperature 99 Cervical Secretions (TDM) 96.5 Symptothermal 99.6 II. Basal Body Temperature Method The basal body t emperature (BBT) rises an initial 0.4 0.8 F during ovulation (Speroff and Darney 1996) due to a surge in luteinizing hormone (Fig. 6) (Germano and Jennings 2006). Stringent record keeping in the form of once daily temperature readings provides a pattern by which peak fertility can be determined. The temperature readings can be taken either orally, vaginally, or rectally prior to getting out of bed in the morning. If temperature elevation is sustained for approximately 3 4 days the woman is assumed to ha ve completed ovulation (Freundl and others 2010). Unlike the calendar method, the BBT method does not account for the number of days a sperm can remain in the uterus and thus recommends abstinence from the beginning of menstruation to the end of ovulation (Pallone and Bergus 2009). Some women choose to use the calendar method to identify additional infertile days prior to ovulation. Similar to the calendar method, factors such as age illness, stress, and extended lack of sleep can decrease the efficacy of this method. Perfect use efficacy estimates of 99% ( Table 5 ) are reported by the World Health Organization (USAID and others 2007). Women may experience a much lower typical use efficacy rate especially when employing the calendar method to estimate "safe sex" days in the first part of her cycle.

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34 III. Cervical Secretion Methods Cervical secretions undergo estrogen induced changes that are highly indicative of fertility. Those changes may incl ude increased mucus, thinner consistency, and increased transparency ( Speroff and Darney 1996) When fertile, cervical secretions tend to be wet, clear, stretchy, and slippery, often compared to egg whites (See Cervical Changes, Day 11 15, Fig 7) Secret ions during infertile periods are comparatively dry, cloudy, and sticky (See Cervical Changes, Day 21 27, Fig. 7) ( Grimes and others 2005; Pallone and Bergus 2009; Freundl and others 2010 ). Several models exist for evaluating the state of the cervical sec retions. One model is called the Billings Ovulation Method which was developed by John Billings in the Figure 7 : Hormonal, follicular, temperature, secretions, and cervical changes during the menstrual cycle Mid cycle secretions have increased mucus whereas late cycle secretions are comparative ly dry (Germano and Jennings 2006).

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35 1970's. The Billings method emphasizes the change in secretion characteristics rather than specific descriptions of each stage (Pallone and Bergus 2009 ). "Safe sex" days occur when little to no mucus is produced (at the beginning of the cycle) until the secretions becomes cloudy and tacky. Over the following 7 14 days, the mucus will increase in volume and become more slippery, stringy, an d clear; marki ng ovulation. Within a few days, the cloudy/tacky consistency returns and coitus can resume until after the next menses ( Schearer 1978 ). It should be noted that the characteristics of cervical secretions is difficult to identify during menses and thus th is period is considered to be fertile (Pallone and Bergus 2009). The Creig hton Model, or NaProTechnology require more active male participation in that the man is encourag ed to maintain and interpret secretion data. This model also provides more standa rdized descriptions of secretion consistencies. The efficacy of the Creighton model in preventing pregnancy is difficult to compare to other cervical secretion methods or even other contraceptives. If a couple has unprotected intercourse on a day that is considered fertile, the Creighton Method indicates that the couple is no longer using the method to prevent pregnancy but to achieve pregnancy. Typical use efficacy rates cannot be compared; however, perfect use efficacy rates should be comparable (Pallo ne and Bergus 2009). The TwoDay Method (TDM) instructs women to check daily for the presence of cervical mucus. If two consecutive days of cervical secretions occurs, the woman is considered fertile regardless of secretion consistency or appearance. Thi s method has a perfect use efficacy rate ( Table 5 ) of 96.5% (Arevalo and others 2002). TDM is simple r than the previous methods because it does not rely on the couple's ability to distinguish be tween secretion cha racteristics which may be ambiguous (Freundl and others 2010). It is not recommended to engage in intercourse on consecutive days. Residual seminal fluid can potentially obscure the state of the cervical mucus ( Speroff and Darney 1996).

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36 IV. Symptotherma l Method The symptothermal method combines two or more of the previously describe d FABMs in order to increase pregnancy prevention efficacy. The perfect use method efficacy ( Table 5 ) can be as high as 99.6% (Pall one and Bergus 2009). Additional symptoms that can be monitored to detect ovulation are mittleschmertz (abdominal pain occurring mid cycle), breast tenderness, and changes in cervix position or texture ( Speroff and Darney 1996). Conclusions FABMs const itute a form of natural family planning that is safe, free of side e ffects, and available to anyone regardless of socioeconomic status or accessibility to alternative methods. There are minimal costs, if any, and FABMs do not require routine physician che ck ups. These methods rely on careful and consistent dail y records over an extended period of time These required tasks are not feasible for many couples The efficacy of FABMs also requires abstinence or the use of secondary contraception during ferti le periods. If a secondary method is not used or if the woman's cycle is irregular, failure rates increase dramatically (Grimes and others 2005). The development of modern methods such as the SDM and the TDM provide a standardized set of rules and guidel ines that may increase acceptability and encourage continued usage.

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37 CHAPTER 7 MALE HORMONAL CONTRACEPTIVES Introduction Male hormonal contraceptive (MHC) development focuses on the inhibition of sperm production (Blithe 2008). MHCs involve the inhibi tion of gonadotropin secretions resulting in a significant decrease in the testicular production of testosterone which consequently inhibits the production of sperm ( Anderson and others 2002; Blithe 200 8 ). Several drug regimens are undergoing Phase III tr ials, yet none have gained approval d espite decades of research. Researchers agree that male contraceptives should feature sufficient spermatogenic suppression, efficacy comparable to that of female contraceptives, ease of use, good tolerability, limited short or long term side effects, and low cost. Contraceptives must be approved by the United States Food and Drug Administration ( US FDA ) and acceptable to the general public (Anderson and Baird 2002; Matthiesson and McLachlan 2006). Background MHCs fo cus on gonadotropin suppression via exogenous steroid administration or the administration of Gonadotropin Releasing Hormone (GnRH) analogs. Progestins, androgens, and GnRH analogs function as contraceptives by intercepting the negative feedback mechanisms at the hypothalamic pituitary gonadal axis and suppressing the production of stimulatory GnRH. L uteinizing H ormone (LH) and F ollicle Stimulating H ormone (FSH) production are consequently inhibited and intratesticular testosterone drops to levels insuffic ient to sustain spermato genesis (Anderson and Baird 2002; Aitken and others 2008 ; Blithe 2008; Grimes and others 2008). A drawback of gonadotropin suppression is that it leads to low concentrations of testosterone in

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38 the serum which negatively impacts the libido and muscle mass and can lead to hypogonadism (Blithe 2008). Contraceptive treatments involve the co administration of an androgen to counteract negative side effects. Androgens have dual functions, either working alone or in tandem with progestin s or GnRH analogs for spermatogenesis suppression. Androgen Only Regimen Androgens, progestins, and GnRH analogs can be administered similarly to many of the current female contraceptives; i.e. orally, implantation, and injection (Blithe 2008). The supp ressive effects of testosterone on gonadotropin secretion and spermatogenesis have been known since the 1930's (Howard and Vest 1939; McCullagh 1939). Testosterone acts at the hypothalamus and pituitary gland to inhibit LH secretion. It also inhibits LH secretion outside of the testes through conversion into estradiol by the enzyme aromatase in the extraglandular tissues (Anderson and Baird 2002). The sole administration of testosterone offers fast gonadotropin suppression as well as androgen replacement, although oral dosing of testosterone is infeasible due t o poor bioavailability (Wu 2006). The following summarizes several androgen only regimens: I. Testosterone Enanthate Weekly intramuscular injections of 200 mg of testosterone enanthate (TE) induc ed azoospermia (<0.1 million sperm/ml semen) in 91% of Asian men and 60% of Caucasian men, and induced oligozoospermia (0.1 million 3.0 million/ml) in the remaining men ( Table 6 ) within a 2 4 month period (Handelsm an and others 1992; Wu 2006). Sperm concentrations of 0 3.0 million/ml resulted in 1.4% chance of pregnancy, which is comparable to female oral contraceptive pills. However, TE injections require frequent and high doses in order to maintain

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39 suppression o f spermatogenesis often leading to acne, weight gain, changes in mood, and decreased high density lipoprotein cholesterol (HDL C) (Wu 2006). Table 6 : Percentage of Asian and Caucasian men reaching oligozoospermia and azoospermia wi thin 4 mo. of weekly TE injections (Handelsman and others 1992; Wu 2006) % Men Affected Race Oligozoospermia Azoospermia Asian 9 91 Caucasian 40 60 II. Testosterone Undecanoate Testosterone undecanoate (TU) is a fat soluble ester (Wu 2006) formulat ed in tea seed or castor oil that is much more stable than TE. TU releases testosterone over a period of 4 8 weeks, prolonging the period between injections. Men in a Chinese study received an initial injection of 1000 mg of TU followed by 500 mg injecti ons in 4 6 week intervals for six months (Gu and others 2003) Azoospermia was reached at about 3.5 months, which is comparable to the length of time needed for TE injections. Side effects included weight gain and a decrease in HDL C (possibly due to the initial large dose of TU). H owever, Gu and others (2003) claim potential benefits, such as increased muscle strength and lean body mass. A phase III multicenter contraceptive efficacy clinical trial was conducted over a period of 30 months by Gu and oth ers (2009) using the same regimen as Gu and others (2003). Results mimicked those of the earlier study (Gu and others 2003) with suppression rates reachi and others 2009). Further testing involving non A sian males would be beneficial to understanding the racial impact on androgen only regimens. Long lasting testosterone preparations that provide a uniform level of spermatogenic suppression would offer significant improvements to the injection method (Wu 2006).

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40 III. Testosterone Implants Pellet implants of fused crystalline testosterone offer similar suppression rates to testosterone injections with fewer hormonal changes and a more consistent level of suppression. Handelsman and others (1992) used pellets containing 200 mg of testosterone that released an average of 9 mg/day. Subjects received six surgically implanted pellets (1200 mg total), which resulted in about 19 weeks of spermatogenic suppression. Data from this study is encouraging but requires testing with larger study groups and more emphasis on potential side effects of long term administration (Handelsman and others 1992). Methyl 19 nortestosterone MENT ( Methyl 19 nortestosterone) is a synthetic androgen that inhibits testosterone synthesis in the prostate but enhances it in the rest of the body. MENT is resistant to 5 reductase, an enzyme that converts testosterone into dihydrotestosterone in the pr ostate. It acts as a substrate for aromatase which converts testosterone to estradiol in the bone (Walton and others 2007). MENT can be used as an androgen replacement therapy in hypogonadal men or as a contraceptive in normal men. It has several key adv antages over testosterone administration, including high potency and rapid absorption. MENT reaches peak blood levels 1 2 hr following an intramuscular injection and is cleared from the body within 24 hr (Prasad and others 2009). Rapid clearance from the body prevents sustained suppression. A sustained release formulation administered by an implant attempts to address this dilemma (Wu 2006). MENT implant combined with progestin treatments aim to further decrease the androgen dose while increasing sperma togenic suppression. Walton and others (2007) found that MENT implants combined with E tonogestrel, a progestin, achieved azoospermia in 8 of 10 men after

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41 12 weeks of treatment. However, spermatogenic suppression was inconsistent, with 50% showing partial recovery at 24 weeks. (Walton and others 2007). Summary of Androgen Regimens Despite advanc es, the androgen only MHC method requires relatively high d oses for adequa te spermatogenic suppression. MENT is 10x as potent as testosterone and is effective at lower doses. However, MENT lacks the ability to maintain long term azoospermia It has been shown that Caucasian men are far less responsive to steroid suppression than Asian men. Diet, genetic, environmental f actors may be responsible for these differ ence s (Wu 2006). Progestin androgen combinations address these problems by reducing the necessary dosage and thereby limiting the hormonal side effects of testosterone alone. Progestin Androgen Combinations Progestins are steroids with receptors in the h ypothalamus and pituitary They are widely used in female hormonal contraceptives and have been more rigorously tested. Progestin administration in men initiates azoospermia via an inhibitory effect on both LH production and FSH secretion. However, prog estins produce several side effects typical of androgen deficiency, including loss of libido. Progestins have been used to augment androgen administration in MHCs (Anderson and Baird 2002). Progestins and androgens work synergistically and allow for lower doses, fewer side effects, and enhanced spermatogenic suppression. Progestin androgen preparations also have wider applications because they can be administered orally in addition to injection and implantation ( Meriggiola and Bremner 1997; Wu 2006).

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42 I. L evonorgestrel Levonorgestrel (LNG) is a commonly used progestin in female contraceptives. It can be used in combination with testosterone injections or implants to enhance the inhibitory effect that testosterone has on spermatogenesis. Testosterone impl ants + LNG have produced azoospermia in 90% of Caucasian men and more than 90% of Chinese men over a 30 week treatment period. The combination treatment has been shown to more effectively suppress spermatogenesis than testosterone injections alone, partic ularly in Caucasian men (Wang and others 2006). More recently, analysis of testicular gene expression following TU + LNG injections has revealed that gonadotropin suppression leads to the upregulation of proapoptotic transcripts Upregulation of transcri pts activates insulin/IGF/relaxin related pathways and limit s the amount of RNA binding proteins. The discovery of testes specific molecules integral to spermatogenic suppression provides potential targets for future male contraceptive research (Lue and o thers 2008). II. Depot medroxyprogesterone acetate Depot medroxyprogesterone acetate (DMPA) is a long acting progestin injected at 3 month intervals that has been used in female contraceptives for several decades (Kaunitz and others 2009). It has been tested in combination with testosterone implants, injections, and gels for effective spermatogenic suppression (Page and others 2006; Amory and others 2007). Testosterone gel (T gel) preparations are more favorable than testosterone injections and exhibit side effects characteristic of testosterone administration, including decreases in HDL, weight gain, and acne The gels can be applied at home and their use decreases the number of injections necessary for the regimen (Amory 2007). T gel + DMPA regimens yielded less than 1

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43 mill ion sperm/ml semen in 80 90% men ages 18 55 yr Testosterone injection and implant combinations produced similar results (Page and others 2006). III. Etonogestrel Etonogestrel (ENG) is a metabolite of the synthetic progestin, desogestrel. It is a more potent progestin than LNG (Wang and others 2006). In 2006, an ENG implant was approved for women by the US FDA ( Anonymous 2006). It has been used in combination with 400 mg testosterone implants (administered every 12 weeks) to induce 100% azoospermia in men. No adverse side effects (i.e. weight gain, lowered HDL C) wer e observed; however, the nine ma n study group was small (Brady and others 2004). Mommers and others (2008) conducted the first large scale placebo controlled d ouble blind study of MHCs on 18 45 yr old men They evaluated the efficacy and side effects of an ENG implant in combination with a TU injection regimen over a 42 44 week period. The ENG implants were inserted in the upper arm and testosterone injections were administered every 10 12 weeks. At 16 weeks, 89% of the study group achieved severe oligozoospermia (less than 1 million sperm/ml) and recovery of previous sperm concentrations occurred 15 weeks following the cessation of treatment. Side effects o f the regimen were typical of current female hormonal contraceptives including night sweats, acne, and weight gain. In sum, the implant injection regimen was found to be effective and reversible, indicating its suitability as an addition to the current market of male contraceptives (Mommers and others 2008). Summary of Androgen Progestin Regimens Androgen Progestin combinations offer enhanced spermatogenic suppression, particularly for Caucasian men who are typically less responsive to androgen only r egimens.

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44 Some side effects typical of androgen administration remain, such as acne and weight gain, but are decidedly less severe than those observed in the androgen only regimens The ENG + TU regimen (Mommers and others 2008) has the most potential for US FDA approval because it successfully suppresses spermatogenesis rates in a large study group. The 2 3 month injection interval provide d significant recovery time, especially when compared with the previously tested weekly injections. However, many in dividuals find injection regimens too invasive for their contraceptive needs. Additional large scale implant or gel studies that support the safety and efficacy of proge stin androgen combinations greatly increase the likelihood of US FDA approval and regi men adherence by patients. G o n adotropin R eleasing H ormone Analogs G o n adotropin R eleasing H ormone (GnRH) analogs are important alternative s to androgen only and androgen progestin regimens because of their swift (within 24 h of administration) spermatogen ic suppression (Wu 2006). GnRH analogs suppress spermatogen esis by interfering with the interaction of GnRH o n gonadotropins prior to gonadotropin secretion (Anderson and Baird 2002). Currently, GnRH analogs are employed in prostate cancer treatment in m en and ovulation regulation in women undergoing in vitro fertilization. Two forms of GnRH analog exist: the GnRH agonist and the GnRH antagonist. suppress ion. GnRH antagonists are competitive blockers of GnRH receptors that offer immediate suppression (Anderson and Baird 2002; Herbst and others 2004). Acyline is a potent, long lasting GnRH antagonist capable of lowering LH, FSH, and testosterone concentra tions to azoospermic levels. A single injection of 300 g/kg resulted in spermatogenic suppression for 15 days, suggesting a contraceptive regimen of twice monthly injections. Side effects were those

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45 normally associated with decreased testosterone levels (Herbst and others 2004). A GnRH antagonist + a ndrogen combination would presumably reduce the risk of hypogonadism similar to progestins. The GnRH agonist, deslorelin, demonstrated spermatogenic suppression in a variety of animals, including opossum s ( Lohr and others 2009), lions (Bertschinger and others 2008), and bats (Metrione and others 2008); however it has not been tested in humans. Current GnRH research focuses on developing an orally active antagonist that will last longer in the body. Previo us GnRH analog formulations had rapid metabolic clearan ce and poor oral bioavailabilty necessitating daily or depot injections. An oral form of the drug would offer more dosage flexibility and it would reduce discomfort normally associated with injections (Ratcliffe and others 2006). GnRH analogs produce high rates of azoospermia in men but they are expensive to produce and have a s hort half life in the body. GnRH analogs could become an appealing fast acting male contraceptive if a viable oral formula could be developed and the manufacturing cost reduced (Wu 2006). Conclusions The addition of MHCs would greatly improve the options available to couples seeking contraception and could reduce the contraceptive burden that many women feel falls more ofte n on them (Martin and others 2000; Wu 2006). Of the regimens mentioned, an androgen progestin combination (i.e. ENG + TU) has the most potential to be approved for Caucasian men, while an androgen only method such as TU is a strong candidate for Asian men They are the only ethnic groups that have undergone phase III multicenter clinical trials for contraceptive efficacy. The androgen progestin combinations may have an even higher chance of approval

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46 because many of the progestins have already been approv ed and marketed for female hormonal contraception. It should be noted that MHC research has been conducted for over 50 years with countless androgen only, androgen progestin combination s and GnRH analog regimens that have proven effective in small and large scale studies. Yet, few have been pursued commercially. Male hormonal contraception appears to suffer from a lack of funding from pharmaceutical companies; however, organizations including the World Health Organization and the Contraceptive Researc h and Development Program continue to support new research, allowing for some progress in the field (Lui 2008). Many proposed regimens must overcome hurdles caused by a poor understanding of gonadotropin roles in germ cell development including inadequate suppression of sperm density, delayed onset of action, and sperm reappearance in ejaculate (Matthiesson and others 2006). Continued research into the mechanisms underlying spermatogenesis would be of great benefit to both the development of male contracep tives and to the treatment of hypogonadism.

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47 CHAPTER 8 MALE NONHORMONAL CONTRACEPTIVES : IMMUNOCONTRACEPTION Introduction Immunocontraception is a proposed form of male and female birth control in which an administered vaccine inhibits conception by acti vating the body's immune response against gametes and/or reproductive hormones (Cooper and Larsen 2006). Sperm atozoa and oocytes contain surface proteins called antigens that are cell specific, immunogenic, and accessible to antibodies. Antibody interact ion with these proteins can lead to the inhibition of sperm binding and fertilization (Suri 2004). Contraceptive Vaccines include three areas of focused inhibition: gamete production, gamete function, and gamete outcome. The inhibition of gamete producti on targets luteinizing hormone releasing hormone / gonadotropin releasing hormone ( LHRH/GnRH ) and FSH. Vaccines that mimick the action of LHRH/GnRH have shown varying degrees of success. GnRH based vaccines are similar to hormonal methods of male contrace ption in that androgen replacement therapy is necessary to maintain libido and secondary sex characteristics. Most L H RH/GnRH vaccines exhibit p artial gametogenesis and sex steroid inhibition (Imboden and others 2006 ; Thau 2006 ) T hey lower hormone produc tion but do no t inhibit it completely T hey currently have lim ited uses in male contraception but are useful when partial inhibition is needed, such as in cases of endometriosis and uterine fibroids (Naz 2009). Vaccines that target gamete production are currently being developed by pharmaceutical companies as an alternative to castration in house pets ( Ladd and others 2003; Wu and others 2009) and mammalian pest s ( Barfield and others 2005; Miller and others 2008) The se vaccines have been shown to be eff ective treatments for prostatic hypertrophy and carcinoma (Suri 2005).

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48 Gamete f unction can be inhibited by sperm antigens and oocyte zona pellucida (ZP). ZP protein based vaccines have also found a niche in animal population control. A common side effe ct in ZP based vaccines is inflammation of the ovaries, oophoritis. H uman applications focus on differentiat ions between infertility related epitomes (B cell epitopes) and oophoritis inducing epitopes (T cell epitopes). Sperm antigens rely on the autoant igenic and isoantigenic potential of spermatozoa ; thus, they can produce antibodies in men and women, initiating an immunological response (Naz 2009). Many sperm antigens are located in the acrosome of the sperm head. During capacitation and the acrosome reaction, the plasma membrane of the sperm head is modified to expose surface proteins allowing for oocyte interaction (Fig. 8 ) (Inoue and others 2005) Figure 8 : Sperm oocyte interaction (1) As the sperm approaches the Zona Pellucida (ZP) it undergoes plasma membrane restructuring to prepare for ZP binding. The sperm head contains an acrosome filled with degradative enzymes. Upon ZP binding, the acrosome expels its contents degrades the jelly coat surrounding the oocyte, and allows the sperm to reach the oocyte in a process called the acrosome reaction (2) (Inoue and others 2005)

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49 Intramuscular injection of autologous or isologous sperm (to men or women), first conducted in women in the 1920's, result ed in the productio n of spermatoxic factors that could prevent pregnancy for up to one year (Baskin 1932; Suri 2004). These spermatoxic factors are now known as antisperm antibodies (ASAs), which are present in ~70% vasectomized men and ~2 30% of infertile couples (Suri 200 4; Naz 2009). ASAs, also known as sperm specific antibodies, are immunoglobulins that bind to sperm antigens and influence fertilization and/or fertility (S uri 2005). The presence of ASA s can lead to infertility through the inhibition of sperm capacitati on, acrosome reaction, sperm zona interaction, and sperm penetration (Naz 2009). Vaccines that target gamete function have a lot of potential due to the natural occurrence of ASAs (Naz and Rowan 2009). Vaccines targeting gamete outcome involve human chor ionic gonadotropin (h CG), a glycoprotein hormone that is secreted by the placenta during pre g nancy (Khademi a nd others 2009). Talwar and others (2009) developed a h CG vaccine that has shown relatively low toxicity and high efficacy. I t is the first contr aceptive vaccine to enter phase I and II c linical trials in humans. The h CG vaccine also has applications in HCG producing cancers. Currently, studies are focus ed on increasing efficac y as well as immunogenicity (Talwar and others 2009). Fertilization Despite the effect of intramuscular spermatozoa injections, the deposition of semen into the female reproductive tract does not produce an antifertility effect. Mammalia n coitus initiates an inflammatory immune response in the cervix and uteru s which resu lts in an influx of neutrophils and macrophages that assist in removing abnormal or non fertilizing sperm. Interaction with seminal factors during this immune response conditions the female reproductive tract for implantation and consequently increases fe rtility rates. Male ejaculate

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50 contains transforming growth factor beta (TGF ), prostaglandins, and other immune regulatory molecules that inhibit paternal specific immune responses initiated by the female's inflammatory immune response. TGF inhibits B cell proliferation and induces a class change of human B cells to IgA, which may favor the production of ASAs in the female reproductive tract to accomplish sperm removal This interaction is highly dependent on the concentrations of t he immune regulatory molecules which differ greatly between ejaculates (Chamley and Clarke 2007) Following deposition, sperm move from the vagina into the cervix. The cervical canal is lined with mucus that assists in guiding healthy sperm into the uterus and blocks the passage of abnormal sperm. The sperm align along the longitudinal axis of muc in fibers in the cervical mucus and swim vectorally. ASAs have been found to interfere with sperm interaction with the cervical mucus by preventing sperm penetration regardless of their normal or abnormal state (Chamley and Clarke 2007). Vaccine Develo pment A ntisperm contraceptive vaccine development begins with the identification of genes and proteins involved in the disruption of fertilization. Disruption targets include sperm production, sperm function and structure, sperm transport and deposition, and sperm eg g interaction (Naz and others 2009). Sperm antigens must contain some degree of specifi city because antigens used for sperm inhibition may also inhibit somatic cells S perm antigen development must include sperm specificity, surface expressi on, fertility involvement, and the ability to raise high titer antibodies that can inhibit fertility (Naz 2009). Gene knock out technology in mice has identified many novel testis and sperm genes and proteins involved in fertility. Mutated genes or prote ins leading to infertility in the mouse can lead to novel

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51 contraceptive vaccine targets in humans. Antigens expressed on the cell surface are most suitable for contraceptive vaccine development because they are available for antibody binding. Proteins Involved in Fertilization Proteins with the potential for vaccine development are identified using gene knockout technology (Naz and ot hers 2009). Knockout mice are bred with the expression of a particular gene inhibited and provide valuable insight into the role played by that gene. Naz and others (2009) reviewed gene knockouts that affect fertility. Most gene knockout affect spermatogenesis directly, although some affect fertilization and even fewer affect mating behaviors. Genes involved in mating b ehavior have little to no application in human contraception, but may be beneficial in managing pest populations. Most gene knockouts that affect spermatogenesis or fertilization provide a nonspe cific route to infertility. Some have side effects includi n g small testes, a predisposition to cancer and severe weight loss. However, several genes/proteins of interest may be worth pursuing for human contraception (Naz and others 2009). I. Izumo One such gene encodes a protein, called Izumo which plays a role in spermatozoa fusing with the oocyte membrane. Female mice immunized with a peptide similar to the Izumo antigen resulted in a 57% reduction in fertility. Izumo mimics also lead to infertility when combined with several other promising antigens in cluding fertilization antigen 1 (FA 1) and testes specific 12 mer YLP 12 (YLP). The combination vaccines produ ced an accumulatory effect and resulted in a 73% fertility reduction rate. All mice regained fertility following cessation of treatment ( Inoue a nd others 2005)

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52 II. Epididymal Protease Inhibitor Eppin (epididymal protease inhibitor) is a protein found in the epididymis and the testes that binds to the surface of human ejaculate spermatozoa and is a new target for contraceptive vaccines. Eppin bou nd spermatozoa entering the ejaculatory ducts encounter the protein semenogelin (Sg) in the seminal fluid, which binds to Eppin and acts as an antimicrobial agent in a coagulum. Immediately following ejaculation, semen liquefaction occurs to free the sper matozoa for movement and fertilization. Sg is cleaved during this period by Prostatic Specific Antigen (PSA), which releases the spermatozoan motility inhibition factor, allowing the spermatozoa to continue with fertilization. When anti Eppin antibodies are added, they block the binding site for Sg. The result is that no coagulum forms and the spermatozoa experience no forward motility, causing infertility. Targeting Eppin for male contraception has so far only been explored in m onkeys, but the effect h as been deemed reversible and further testing is underway (O'Rand et al 2006). Overall, contraceptive vaccines have limited adverse effects, high specificity, and require infrequent admin istration. H owever, protective immune response varies in the indivi dual, making it difficult to predict an average immune response and likewise a generic effective dose (Naz and Rowan 2009). III. Lactate Dehydrogenase C 4 Lactate dehydrogenase C 4 (LDH C 4 ) is a sperm specific enzyme (Odet and others 2008) important in s perm energy metabolism and capacitation. It is expressed during primary spermatocyte development in mammals and is highly immunogenic. The LDH C 4 immune response leads to infertility by reducing the sperm swimming capacity and by causing head to head agg lutination as evidenced by immunnohistochemical results (Goldberg and others 2010)

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53 An immune response can be initiated by a nucleic acid vaccine containing a recombinant plasmid with genes encoding human LDH C 4 Nucleic acid vaccines (DNA and RNA) foste r several advantages over attenuated and genetically engineered vaccines T hey can induce a humoral and cellular immune response, the delivered antigen protein is identical to the native antigen, and they are not pathogenic. The LDH C 4 DNA vaccine can be created by inserting the LDH C 4 gene into an expression vector. Yong and others (2008) inserted amplified mouse LDH C 4 coding sequence into the eukaryotic expression vector pVAX1 and transformed it in E. coli immunization of female and male mice Mucosal immunization resulted in a higher antibody titre than intramuscular injection and stimulated the local and systemic humoral immune responses to a higher degree in male mic e than female mice (Yong and others 2008 ). IV. Fertilization Ant i gen 1 Fertilization Antigen 1 ( FA 1 ) is a sperm specific antigen involved in human immunoinfertility (Menge and others 1999) Induced FA 1 antibodies inhibit sperm capacitation and the acrosome reaction by inhibiting phosphorylation of threonine, tyrosine, and serine residues C onsequently sperm egg binding is also reduced Female mice immunized with a FA 1 DNA vaccine exhibited 44 70% reduction in fertility. C ombination vaccines which include FA 1 and YLP have shown enhanced rates of infertility when compa red with FA 1 vaccines alone but more research is necessary to confirm the results (Naz 2006). V. YLP 12 YLP 12 (YLP) is located in the acrosome of spermatozoa and participates in sperm zona pellucida interaction (Choudhury and others 2009), capacitatio n, and the acrosome reaction. YLP DNA vaccine d female mice produced long term reversible contraception one year after

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54 treatment cessation (Naz and Chauhan 2002). YLP has also been delivered to female mice via virus li ke particles (VLPs) to produce ~40% r eduction in fertility, although the rate may be improved by the addition of an adjuvant (Choudhur y and others 2009). VI. Cation Channel of Sperm Protein Family Cation channel of sperm (CatSper), a protein family expressed in the testes (Avenarius and othe rs 2009) plays a role in sperm hyperactivity and male fertility. Knockout mice deficit in any one of the CatSper members are completely infertile due to impaired sperm mobility. CatSper proteins effect sperm ion channels and are exclusively expressed by spermatogenic cells (Li and others 2009) CatSper specificity and effect on sperm mobility lends belief that the CatSper family would be an ideal immunocontraceptive target (Li and others 2009). Future Prospects The maximum fertility reduction reached with sperm antigens in the mouse is ~75% (O'Rand and others 2006) far below the perfect use efficacy rate for hormonal contraceptives, condoms, and vasectomy; however, limitations could lie in the animal model used (Naz 2009). Current research focuses o n finding new targets for contraceptive vaccines and improving the methodology already developed. Immunocontraception would benefit from improvements in B cell epitope vaccines, multiantigens, immunization pathways, and vaccine delivery vectors. Further knowledge of antisperm antibodies (ASAs) and how they interact with sperm antigens may lead to novel antigenic targets for contraceptive vaccines It should be noted that ASAs are not co nfined to infertile individuals. They are found in a small percentag e

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55 of fertile men and women, suggesting that although their presence is associ ated with infertility, ASAs may not directly disrupt fertility (Chamley and Clarke 2007). Immunocontraception strategies will most likely incorporate DNA/RNA vaccine delivery due to its aforementioned advantages. New developments in sperm proteomics and knockout genes using RNA interference could assist in the identification of additional sperm specific proteins. Further, newly identified sperm proteins could provide better unde rstanding of the ligand interaction using X RAY/NMR techniques and elucidating the structural biology of sperm proteins (Suri 2004). Conclusions Immunocontraceptives have several advantages i n the male contraceptive field They offer long acting, reversible, and easy to administer contraception, the means for which have already been implemented in most countries due to disease vaccine delivery. A sperm or testes specific antigen can isolate the immune response to the reproductive tract and avoid unnecessary side effects to the rest of the body (Aitken 2002; Li and others 2009). The antisperm vacci ne strategy works best in women because it targets the relatively small number of spermatozoa fo und in the upper female reproductive tract post ejaculation. Male directed vaccines must target the much larger spermatozoa population found in the male reproduction tract pre ejaculation (Suri 2005). C ontraceptive vaccines must exhibit a high degree of reliability as well as reversibility. Human beings have considerably diverse genomes and likewise have a relatively wide range of immunological responses to various antigens. The infertility effects produced by an antigen in a small subset of individual s may not be universal (Suri 2004) S ome form of antibody monitoring system may be necessary to ensure continuous immunological response in

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56 the individual This would likely increas e the cost and therefore decreas e its suitability for low income couples. R eversibility is an important consideration for all forms of birth control. Variability in immune response leads to variability in the length of time a contraceptive is effective Infertility that lasts for unknown periods of time is useless to indivi duals seeking contraception as a form of family planning. The long term effects of contraceptive vaccines must also be evaluated in humans. Knowing the details of when the contraceptive effects will be reversed as well as the length of time the method ca n be safely used are important consideration s for many couples. It is possible for contraceptive vaccines to be used as an alternative to surgical sterilization by delivering an immunotoxin that would destroy sp ermatogonial stem cells. A site specific ta rget would preserve male behavior and libido while ensuring permanent infertility (Aitken 2002). Permanent i mmunocontrac eption is a realistic alternative to castration in pets and mammalian pests It could prov ide a less invasive and more cost effective method of sterilization although some considerations must be made Immunocontraceptive use in wild or feral animals may lead to genetically based nonresponse to contraceptive vaccines on the ge ne pool There will inevitably be a small group o f the target population that will be resistant to the vaccine Resistant individuals will retain the ability to reproduce. This could lead to the artificial selection of non responders which could potentially negatively impact the immunogenetic c onstitu tion of the population (Cooper and Larsen 2006). H umans can avoid this dilemma by shear availability of alternative contraceptiv es and by their ability to control their own fertility rate, but it is important to maintain a good grasp on long term, overall eff ects

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57 CHAPTER 9 MALE NONHORMONAL CONTRACEPTIVES: PLANT DERIVED COMPOUNDS AND INDAZOLE CARBOXYLIC ACIDS Introduction Nonhormonal contraceptives have some a dvantages over hormonal methods. They act quickly and they do not lead to hormonal imbalan ces outside of the reproductive tissues (Anderson and Baird 2002). Plant derived contraceptive compounds are commonly utilized in traditional folk medicine and are often c with the implication of fewer si de effects. This is a common misconception in many countries and religious groups, but plant derived compounds may be more ac ceptable to the male contraceptive user population due to these beliefs Several notable plant derived compounds include gossypol (extracted from cotton), Tripterygium wilfordii Hook, and Aza dirachta indica A. Juss. Indazole carboxylic acids are a class of compounds that nonhormonally target Sertoli cell germ cell adhesion, leading to reversible infertility. Adjudin is an indazole carboxylic acid that is particularly noteworthy Numerous groups of nonhormonal contraceptives are in research trials; however, an extensive review is beyond the scope of this thesis. I. Gossypol Extract of Cotton The contraceptive effects of gossypol have been known since the 1950's (Liu 1957) Researchers found that cooking with a crude cottonseed oil instead of soybean oil produced a decade of infertility in Wang V illage in Jiangsu, China ( Liu 1957; Qian and Wang 1984) Gossypol is a polyphenolic c ompound (Fig 9 ) derived from the seed, stem, and roots of cotton plants (Wang and othe rs 2009). Gossypol inhibits testicular lactate dehydrogenase (LDH) by directly

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58 competing with NADH for a binding site on LDH (Patel and others 2008). This leads to de creased spermatogenesis and decreased post ejaculatory sperm motility, without affecting Leydig cell function (Mruk and Cheng 2008; Anderson and Baird 2002). A study involving over 8000 Chinese men found gossypol administration resulted in 90% oligospermi a H owever undesirable side effects included hypok a lemia (periodic paralysis) and irreversibility in ~20% of users These effects discouraged further research despite indications that the side effects may have been dose dependent (Zavos and Zarmakoupis Z avos 1996; Roychoudhury and others 2009). Little gossypol research exist s today; however, it was found to inhibit Bcl 2 family of proteins while inducing proapop totic proteins making it useful in cancer treatments ( Priyadarshi and others 2010) Researche rs have also investigated gossypol's anti amoebic (Gonzalez Garza and others 1989), anti malarial (Gomez and others 1997; Patel and others 2008), and anti HIV (Polsky and others 1989; Royer and others 1991) characteristics. Figure 9 : Molecular structure of Gossypol (Shaddack 2007) II. Tripterygium Thunder God Vine, Tripterygium wilfordii is a woody vine found in Eastern and Southern China, Korea, Japan, and Taiwan (Brinker and others 2007) It is used in traditional Chinese medicine in the form of a hot water ethyl acetate, or chloroform methanol extract s to treat

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59 inflammation, sores, and fever (Tao and Lipsky 2000; Brinker and others 2007 ). Tripterygium extracts w ere first noted by Yu ( 1983 ) while conducting research on th e extract's treatment of rheumatoid arthritis. Tripterygium used for the treatment of rheumatoid arthritis and psoriasis at 20 30 mg result ed in azoospermia or non motile sperm within 2 months (Lue and others 1998). Tripterygium extract is a mixt ure of s everal active compounds including triptolide (Fig 10 ) and tripdiolide (Fig. 1 1 ) Both compounds induce azoospermia in rats (Zhen and others 1995). The precise mechanism for sterility is unknown; however research indicates that they may inhibit Ca 2+ cha nnel s (Bai and Shi 2002) or alter spermiogenic gene expression (Brinker and others 2007). Tripterygium trea tment is not associated with adverse effects and does not affect levels of LH, FSH, or testosterone (Qian and others 1988). Despite the use of T rip terygium derivatives in a variety of rheumatoid arthritis and skin disorder therapies, research focusing on the anti fertility effect in Ye 1991; Matlin and others 1993) are rare within the last 10 years. III. Azadirachta indica Neem, Azadirachta indica A. Juss is a flowering tree native to India and Southeast Asia that has been used for centuries in traditional Ayurvedic medicine in India. Many parts of the Figure 11 : Molecular structure of triptolide (Kuebelbeck 2008) Figure 10 : Molecular structure of tripdiolide (Kuebelbeck 2008)

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60 Neem tree have been u s ed in the traditional medicine including the bark, leaves, fruit, root, and seed. Further, over 140 compounds have been isolated from neem some with spermicidal properties: nimbidin (seed oil extract), nimbin (seed oil extract), and salanin (fruit pulp e xtract) (Brahmachari 2004). Neem leaf extracts showed reversible spermicidal activity in vitro (Khillare and Shrivastav 2003) and antifertility activity when administered orally to mice as a fresh green leaf extract (Deshpande and others 1980) and as a dr ied leaf extract mixed with water (Aladakatti and others 2001). IV. Indazole Carboxylic Acids Indazole carboxylic acids are a class of compounds that target germ cell adhesion to Sertoli Cells (Mruk 2008). Lo nidamine and adjudin are indazole carboxyl ic acid analogs that produce infertility in mammals. Lonidamine results in germ cell depletion in the seminiferous epithelium but the effect is associated with irreversibility, hepatotoxicity, and nephrotoxicity (Mruk and others 2008). Adjudin, 1 (2,4) dichlorobenzyl 1 H indazole 3 c arbohydrazide (Fig. 1 2 ) acts on specific adherens junctions in the testis known as apical ectoplasmic specializations (apical ES). Adjudin leads to infertility by disrupting the connection between the Sertoli cells of the se miniferous epithelium, and round and elongate spermatids (Kopera and others 2009). Previously, it had been reported that oral administration of Adjudin (50 mg/ kg of body weight ) resulted in liver inflammation and muscle atrophy (Grima and others 2001). A djudin ( 25 mg/kg body weight ) administered to male Japanese rabbit s produced no such side effects. H owever, oral administration had poor bioavailability, possibly due to poor absorption or rapid metabolism in the gastrointestinal tract (Hu and others 2009 ). Mruk and others (2006) reported the use of a mutant FSH protein carrier to deliver Adjudin specifically to the testis resulted in increased efficacy and decreased toxici ty The Adjudin FSH mutant protein conjugate induce d

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61 infertility at a lower dose ( kg of body weight ) (M ruk and others 2006). The mutant protein has high production costs and must be delivered intramuscularly, one of the least acceptable contraceptive delivery methods (Mruk 2008) Finally, long term exposure to the Adjudin FSH m u tant has not been investigated but remains a concern due to the high level of tissue restructuring in the testis (Mruk 2008). Conclusions Non hormonal male contraceptives have a variety of advantages but are hampered by limited understanding of the mol ecular regulation of spermatogenesis. Recent advances in genomics, proteomics, and gene knockout experiments have identifi ed numerous proteins and genes integral to spermatogenesis. Crystallographic and RNAi studies have been useful in exploring genes fu nction and protein function C ontinuation of such studies w ill support the growing experimental field of non hormonal male contraceptives and may lead to a marketable product. Figure 12 : Mol ecular structure of adjudin (Meodipt 2009)

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62 CHAPTER 10 CONCLUSIONS The introduction of a male contraceptive pill, gel, or injection regimen poses several societally relevant issues. Hormonal and non hormonal contraceptives do not offer protection against sexually transmitted infections (STIs); therefore, the male and female condom would not be rendered obsolete. Men who are not in disease free monogamous relationships would be well advised to continue using condoms as part of their disease prevention regimen. In addition, many couples rely on two methods of birth control (ex. a male condom and a female hormonal contracep tive). It is unclear whether couples would employ both the condom and male hormonal contraception in the same manner. It is very important to initiate STI education targeting individuals considering non barrier male contraception; particularly young adul ts. Although these topics arise when proposing the use of non barrier male contraceptives it is clear that additional easy to use, effective male contraceptio n would be valuable addition s to the available birth control methods. Hormonal and nonhormonal male contraceptives face several obstacles before regulatory approval can be granted. As mentioned previously, contraceptives must undergo rigorous safety testing in which high efficacy is demonstrated and side effects are limited (or non existent) in or der to be acceptable for long term human use. Decades of research have been devoted to male contraceptive development, yet the scientific community has not found a male contraceptive that is comparable to the female birth control pill. Advancements in male directed contraception have been limited to alternative condom materials and vasectomy techniques, whereas female contraceptives have seen numerous advancements in the methods of delivery and the reduction of side effects (Anderson and Baird

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63 2002; Ait ken and others 2008; Rowlands 2008). Researchers have proposed a wide range of reasoning behind this divide but the majority of researchers agree that there is a perceived lack of need, particularly by pharmaceutical companies. Pharmaceutical support of male contraceptive development would be beneficial in encouraging reproductive scientists t o pursue contraceptive research. It m ay also increase private and public funding available to contraceptive researchers. Such undertakings have been attempted in recent years. For example, European pharmaceutical companies Schering AG (acquired by Bayer in 2006) and Organon (acquired by Merck in 2009) collaborated on male contraceptive research in 2002. They expected to have a marketable male contraceptive withi n 5 7 years; however, their partnership ended in 2006 due to a change in business organization, the need for continued clinical trials, and the perception that too few men would be interested in an implant or injection method of birth control (Oaks 2009). In contrast to this perception Martin and others (2000) have shown that injection/implant acceptability may vary by region. Male survey participants from the U.K. ranked implants as the least acceptable method while the pill was ranked the most acceptab le method. On the other hand, male survey participants in China ranked implants as the second most acceptable method, while the pill was the least acceptable. The two methods were also compared to condoms and a three monthly inj ection (Martin and others 2000). S urveys have also shown that up to 83% of men (Martin and others 2000) and 71% of women (Glasier and others 2000) support the idea of a male hormonal contraceptive. The etonogestrel + testosterone undecanoate (progestin androgen) regimen and the testosterone undecanoate (androgen only) regimen have both reached phase III clinical trials and are the most promising hormonal meth ods for men. Many of the hormonal and nonhormonal methods

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64 described previously have poor bioavailability orally; therefor formulate. Injection and implant regimens are much more likely. The perceived need for m ale contraception is further supported by the belief that the contraceptive burden falls on women too often (Glasier and others 2000). Family planning efforts have been primarily female directed, perhaps because of a woman's vested interest in preventing pregnancy. Alternative male methods may contribute to a shift in contraceptive burden from women to men or couples. Summary T he male contraceptive research field has produced several promising methods. Surveys have shown a pote ntial market for barrier, oral, injectable, and implantable male contraceptives. Because non barrier contraceptives provide no protection against STI s, novel methods should target men in long term monogamous relationships. Finally, male contraceptive choice has consequences for both men and women. The decision to use a novel male contraceptive should therefore be discussed by both partners to evaluat e the risks and benefits for each method.

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