EP0209590A1 - The use of male essence, including androstenol and dehydroepiandrosterone sulfate to treat luteal phase defects and failure to ovulate in human females - Google Patents

Abstract

A new method of modifying the duration of the menstrual cycle in women includes exposing a woman's nasal region to at least some of a man's axillary secretions. This exposure continues in amounts and for a duration effective to significantly modify the expected duration of the cycle of this woman. In particular, the parts of the male axillary secretions to which the woman's nasal region must be exposed preferably comprise one or more compounds such as androstenol, dehydroepiandrosterone and / or one or more compounds having a heavy and burnt odor, similar to the axillary odor, eluted in approximately 50 minutes, as illustrated by the response curve of the flame ionization detector (FID) of FIG. 3B. Studies show that secretions collected from male donors are effective in altering aberrant menstrual cycles, especially those with lutein phase defects, so that they become presumed fertile menstrual cycles lasting 29 , Approximately 5 days, with a variation of 9E 3 days.

Description

THE USE OF MALE ESSENCE, INCLUDING ANDROSTENOL AND DEHYDROEPIANDROSTERONE SULFATE TO TREAT LUTEAL PHASE DEFECTS AND FAILURE TO OVULATE IN HUMAN FEMALES

Background of the Invention The present invention relates to the field of reproductive biology, and more particularly to methods used by reproductive biologists, gynecolo¬ gists and/or obstetricians to predict in human females the probability that a given menstrual cycle type is presumptively fertile or infertile, the timing of the menstrual cycle and/or the onset of the fertile period, the time of ovulation. The field of the present invention includes the field of detection and diagnosis of ovulation in human females through the detection of secondary characteristics occurring during or at the time of ovulation, and more particularly, to the identification and detection of such secondary characteristics as they appear in human females. For many years there has been a need to detect and diagnose the precise time of ovulation in a given female mammal. It can be of great importance, for example, to pinpoint the time of ovulation to ensure that fertilization occurs or to prevent conception. Alternatively, it may be important for other medical reasons to diagnose ovulation.

To some extent, methods of diagnosing ovulation are disclosed in the above-identified related patents. Additionally, the occurrence of ovulation can be established with some certainty through various prior art methods. For a review of many of the known surgical, clinical, biochemical or hystological techniques for diagnosing ovulation, please refer to the descriptions appearing in the above-mentioned related patents and patent applications, particularly to columns 1-5 of U.S. Patent 4,119,089.

Perhaps the most popular and widely used method of estimating the time of ovulation relies upon the graphic recording of the waking temperature at basal conditions (hereinafter referred to as the BBT method) . Using this method a dedicated woman with uniform daily habits can determine the time of Ovulation within two days after its occurrence. In recording the basal body temperature, a rise in temperature is commonly associated with the beginning of the luteal phase, but can vary from the actual time of ovulation by as much as 72 hours. Figure 1 of U.S. Patent 4,119,089 illustrates a theoretical basal body temperature chart showing a biphasic cycle having a lowered body temperature during the follicular phase, and a sustained raised body temperature during the luteal phase. Another technique which is commonly used to determine the time of ovulation in human females is the charting of concentrations of certain hormones appearing in the blood. In humans, a preovulatory rise in serum estrogens coupled with a sharp rise in luteinizing hormone (LH) levels as determined by radio-im unoassay of serially drawn blood samples, is perhaps the most accurate indicator of impending ovulation. Ovulation most likely occurs 12-24 hours after maximum LH levels. A subsequent persistent in- crease in levels of serum progesterone indicates that ovulation has occurred. Since these determinations are expensive and not widely available, clinical parameters such as BBT parameters are most often used to determine the time of ovulation.

The field of the present invention also relates to methods of predicting the onset of the fertile period. It is generally accepted that the maximum survival function of spermatozoa capable of fertilizing an ovum is approximately three days following coitus. Although theoretically any coitus prior to ovulation entails a certain risk of pregnancy, as a practical matter, abstinence from un¬ protected sexual intercourse for at least three days (preferably up to five days) prior to ovulation is generally considered sufficient to avoid pregnancy. It is generally recognized that a human ovum is (in vivo, fertilizable for about 12 hours and certainly for no more than 1 day following ovulation. The human fertile period, then, is made up of no more than 4 to 6 days out of the entire menstrual cycle. If it were possible to accurately predict this fertile period, in order to avoid pregnancy it would only be necessary to abstain from unprotected intercourse or use alternate birth control methods during that 4-6 day "fertile period" rather than for the entire menstrual cycle. Notwithstanding the methods of the aforementioned related patents, the only technique yet widely used for "predicting" the fertile period of a female is the method which relies upon basal body temperature determination of ovulation in a plurality of preceding cycles to determine the expected time of ovulation for future cycles. This method is not really directed to ascertaining the precise fertile period for a given cycle, but rather is intended to identify a period during which coitus is statistically likely to produce pregnancy. Based on calendar records of 21,499 menstrual cycles experienced by 592 healthy women living in Switzerland, 9.7% of all menstrual cycles range in length from 6-23 days, 80.1% into the range from 24-34 days, and another 10.2% cover the remainder of menstrual cycles from 35 to 409 days in length. The modal cycle length of the cycle of 28 days duration and the average cycle length amounts to 29.2 days. See "The Degree of Variability in the Length of the Menstrual Cycle in Correlation with Age of Woman", by R.F. Vollman, Gvnaecoloσia. • 142(5) ; 310-314 (November 1956) . Since this information is based upon past performance, and since the time of ovulation varies markedly between different individuals as well between cycles of a given individual, the period for abstinence must be long enough to considerably reduce the possibility of pregnancy. As explained in U.S. Patent 4,119,089, taking into account the variability in menstrual cycle length, even the most regular women probably need to abstain from unprotected coitus for more than one-third of the total menstrual cycle in order to avoid pregnancy. Accordingly, a long felt need exists for methods of more accurately predicting or detecting the onset of the fertile period for a given cycle, such that coitus can be avoided, or other contraceptive methods employed, during the fertile period of that cycle.

The present invention also relates to the field of mammalian chemical communication, and more particularly to research involving mammalian pheromones. It has long been known that the estrus cycles of certain mammalian species are effected by pheromones and that their estrus cycles may be artificially manipulated. For examples, estrus cycles of rats can be manipulated through the use of odors collected from females during specific phases of the estrous cycle. See McClintock, M.K., "Pheromonal Regulation of the Ovarian Cycle: Enhancement, Suppression and Synchrony", Pheromones and Reproduction in Mammals, edited by J.G. Vandenbergh, New York: Academic Press, pp. 113-149 (1983) and McClintock, M.K., "Estrous Synchrony: Modulation of Ovarian Cycle Length by Female Pheromones", Phvsiol. Behav. 32.:701-705. In addition, preovulatory cervical mucus mixed with water and sprayed into the noses of a group of female Holstein cows advanced and synchronized the time of estrous. See Izard, M.K., "Pheromones and Reproduction in Domestic Animals", Pheromones and Reproduction in Mammals, edited by J.G. Vandenbergh, New York: Academic Press, pp. 253-281 (1983) and Izard, M.K. and Vandenbergh, J.G., "Priming Pheromones From Oestrous Cows Increase Synchronization of Oestrus in Dairy Heifers After PGF-2 Injection", J. Reprod. Fert. 66:189-196 (1982). Notwithstanding these and other reports of elaborate pheromonal systems in other mammals there has been no experimental evidence that pheromonal systems operate in humans.

More recently a number of studies have suggested the possibility that human odors act in a manner analogous to primer pheromones in animals and alter reproductive endocrinology. Although anecdotal reports of aphrodesiacs are as old as modern civilization, the possibility that human reproductive biology can be altered by pheromones was not considered seriously until McClintock published a report in 1971 that menstrual synchrony occurred among certain women attending a predominantly female university. In "Menstrual Synchrony and Suppression", Nature, 221:244-245 (January 22, 1971) McClintock reported that "social interaction" can have a strong effect on the menstrual cycle of women living together in a college dormitory. The McClintock study compared the dates of menstrual onset for roommates and close friends, and for living groups. McClintock reported that a signficant increase in menstrual synchrony was found among roommates, among closest friends, and among roommates and closest friends combined.

McClintock hypothesized that the synchrony could be due to some factor other than time spent with an individual such as the available food and/or synchrony of the moon. McClintock further speculated that synchrony might parallel the Whitten effect in mice in which suppression of oestrous in groups of females may be released by the introduction of a male mouse pheromone. McClintock suggested that synchrony might result from a pheromonal interaction of suppression among close friend groups, followed by a periodic release due to the presence of males on the weekend.

McClintock stated:

"However, this would be insufficient to explain the synchrony which occurred among roommates and close friends but did.not occur throughout the dormitory."

A further possible explanation advanced by McClintock was the awareness of menstrual cycles among friends, however a sample taken from the dormitory indicated that 47% were not conscious of their friends' menstrual cycles, and of the 53% who were, 48% were only vaguely aware. While McClintock was able to conclude that a significant factor in synchrony is that individuals in the group spend time together, McClintock was ultimately unable to state whether the mechanism underlying this phenomenon is pheromonal, mediated by awareness, or some other process, indicating that the question "still remains open for speculation and investigation". McClintock also explained:

"Exposure to males may not be the significant factor. It may be, for.example, that those with longer cycles are less likely to spend time with males. However, many subjects spontaneously indicated that they become more regular and had shorter cycles when they dated more often. For example, one subject reported that she had a cycle length of six months until she began seeing males more frequently. Her cycle length then shortened to 4.5 weeks. Then, when she stopped seeing males as often, her cycle lengthened again. Whether this is due to a pheromone mechanism similar to the Lee-Boot effect in mice has yet to be determined." [Lee and Boot, Acta physiol. Pharmacol. Neerl. _f 5, 213 (1956)]. Subsequent investigators have also considered the effect on menstrual synchrony of females in different living conditions. In "Menstrual Synchrony in Female Undergraduates Living on a Coeducational Campus", Psychoneuroendocrinology, -?245-252 (1980), Graham and McGrew report investiga¬ tions of menstrual synchrony among female undergraduates living on a coeducational university campus. A significant trend toward synchrony was found for closest friends, but no significant effect occurred for neighbors or randomized pairs. Nor did any significant correlation emerge between cycle length, duration, duration of menstruation, or the amount and nature of social interaction with males. Graha-m and McGrew accordingly conclude that the amount of time individuals spend together, not similar living conditions, is the significant fact in synchrony. Graham and McGrew further conclude that the "how and why" of menstrual synchrony remain unknown, indicating that, as suggested by Rogal in "A Critical Evaluation of the Possibility of Higher Primate Reproductive and Sexual Pheromones", Psychol. Bull. 85:810-830 (1978), menstrual synchrony cannot be explained by any existing hypothesis other than a pheromonal one.

In 1981, Quadagno et al reported a tendency toward menstrual synchrony which was greatest between roommates and close friends. See Quadagno, D.M., Shubeita, H.E., Deck J.^' and Francoeur, D. , "Influence of Male Social Contacts, Exercise and All Female Living Conditions on the Menstrual Cycle", Psychoneuroendocrinol. 6_:239-244 (1981). Casual exercise performed regularly was associated with longer menstrual cycles whereas spending "social time" with a male was reported not to have any effect on the length of the menstrual cycle for the women in the study. Noting McClintock's finding that "women seeing males less than 3 times a week experience significant¬ ly longer cycles than women who spent time with males more than 3 times per week", Quadagno nonetheless confirms Graham and McGrew's findings that no signifi¬ cant correlation exists between cycle length and the -amount and nature of social interaction with males.

Most recently, Veith et al have reported that exposure to men has the capacity to shorten the menstrual cycle in women. See "Exposure to Men Influences the Occurrence of Ovulation in Woman", Physiology and Behavior 31:313-315 (1983). Veith et al report that women who spent two or more nights with men during a 40 day period exhibited a significantly higher rate of ovulation as determined by basal body temperature charts than those spending no or one nights. Cycle length was not effected by sleeping arrangements, and the frequency of sexual intercourse was said to be unrelated to either cycle length or likelihood of ovulation. The mechanism underlying this phenomenon was reported to be "unknown", but was "conjectured" to be pheromonal in nature. Veith et al thus conclude that "a significant variable contributing to the likelihood of ovulation are the number of nights a woman spends in the same bed with a man". Veith et al further conclude that cycle length "was not influenced by this factor", noting that the

"lack of replication of the earlier [McClintock] study concerning the lengthening of cycles in those women with limited exposure to males is surprising". Veith suggests that the naturalistic conditions resulting from subjects being enrolled in a coeducational institution may provide participants with sufficient exposure to men so that their cycle length would not be unduly increased.

A number of studies have investigated the possible effect of underarm perspiration on menstrual synchony. In 1977, Russell, Switz and Thompson reported on the effects of using the female axillary sweat of a single, so-called "driver" female as a stimulus towards menstrual synchrony. See "Olfactory ^• Influences on the Human Menstrual Cycle" presented at the meeting of the American Association for Advance¬ ment of Science, San Fransisco, June 1977 and published in Pharmac. Biochem. Behav. 13.(5) :l-2 (1980) . Russell et al had female volunteers rub on their upper lips cotton pads containing the perspira- tion odor of a particular donor female. After four months the menstrual onset dates of the volunteers were significantly closer to that of donor female than were the cycles of the control group who received the application but not the odor. The particular female donor subject had a history of a very regular menstrual cycle of 28 days and no significant history of menstrual problems. She had demonstrated a previous experience of "driving" another woman's menstrual cycle on three separate occasions over three consecutive years, did not use underarm deodorant nor shave under her arms. These data showed that the mean difference from onset of the menstrual cycle of the subjects from the donor was 9.3 days in the pretreat- ment month and 3.4 days in the posttreatment month for the experimental group, and that this change showed statistical significance over the control group. Russell et al thus conclude that "odors from one woman may influence the menstrual cycle of another and that these odors can be collected from the underarm, stored as frozen samples, for at least short periods, and placed on another woman." The experiment was further said to support the theory that odor is a communicative element in human menstrual synchrony, and that at least a rudimentary form of olfactory control of the hormonal system is occurring in humans in a similar, fashion to that found in other mammals.

As Russell et al noted, it is possible that volatile substances are transferred to the nose even though the subject has no awareness of them. It is also possible that the mechanism of transfer in the Russel et al study did not involve the nose at all, but diffusion of chemical compounds through the skin which may occur when the sample was placed on the subject's upper lip.

Underarm sweat comprises secretions of the apocrine, eccrine and sebaceous glands. Analyses of apocrine secretion have shown the presence of protein (10%), cholesterol (1%) and two androgen steroids: androsterone sulfate and dehydroepiandrosterone sulfate (0.2%). The apocrine secretion as collected at the skin surface is odorless; however, incubation with the resident skin bacteria results in an odor profile unique to that organism. The micrococci bacteria give an "acid odor" to the secretion which has been characterized by head space analysis as isovoleric acid. See Labows, J.N., "Human odors - what can they tell us?". Perfumer and Flavorist 4.:12-17 (1979). The diptheroid bacteria give a similar head space profile but the observed odor is the more distinct "apocrine (human) odor" usually associated with the axillary areas.

Similar correlations of odor quality and bacterial populations were found in vivo. The axillary miσroflora of 229 subjects have been characterized quantitatively and their results correlated with the type of donor found. Micro- coccaceae were present in all subjects and were the dominant organism when a faint or acid odor was noted. The aerobic diptheroids were found in 85% of the males and 66% of the females and were associated with a pungent, apocrine odor. There were no significant differences related to handedness or presence of axillary hair. See Leyden, J.J., McGinley, K.J. Hoelzle, E., Labows, J.N. and Kligman, A.M., "The Microbiology of the Human Axillae and its Relation to Axillary Odors", J. Invest. Dermatol. 77:413-416 (1981). The "apocrine odor" is similar to that of androst-16-en-3-one. See Labows, J.N. 1979, supra. Neither this steroid nor androst-16-en-3-ol, a musky odorant are present in orginal odorless secretion but they have been -shown to be present in axillary sweat. See Bird, S. and Gower, D.B., "Axillary 5 -androst-16- en-3-one. Cholesterol and Squalene in Men: Preliminary Evidence for 5 -androst-16-en-3-one Being a Product of Bacterial Action", J. Steroid Biochem. 12:517-522 (1982); Bird, S. and Gower, D.B., "Measurement of 5 -androst-16-en-one in Human Axillary Secretions by Radioimmunoassay", J. Endocrinol. j$5_:80-90; and Brooksbank, B.W.L., Brown, R. and Gustafsson, J.A., "The Detection of 5 -androst-16- en-3-ol in Human Male Axillary Sweat", Experientia 3_0:864-865 (1974). In experiments in which axillary bacteria where incubated with sterile apocrine sweat, typical pungent malodor was produced only with aerobic diptheroids. This included both isovoleric acid and another component which was pungent. The odor is similar in character to androst-16-en-3-one and this material has been detected on axillary pads using radio-immunoassay procedures. See Bird, S. and Gower D.B. supra. Studies of extracts of odorous cultures of diptheroids grown on apocrine secretion by GC/MS as yet have not revealed detectable levels of androstenone. It is reasonable to assume, however, that further studies will reveal them since they are detected in axillary washings. See Personal Communication, 1984. Dr. J.N. Labows, Monell Chemical Senses Center. Apart from the above-mentioned data, Graham et al describe the remaining evidence for pheromonal communication of a human female's hormonal status as being "largely circumstantial and fragmentary". For example, Graham et al cite work on short-chain fatty acids in human vaginal secretions; vaginal odors; odor discrimination studies; effects of alleged human pheromonal preparations on subject performance; and scent-marking experiments in public places using androstenone, a mammalian pheromone, which is said to alter spontaneous behavior. See for example, Clark, Klassnick and Watson, "Human Responses to Scent- Marking with the Putative Pheromone Androstenone" (1978) (unpublished manuscript cited in Graham et al supra) . in addition to the above, numerous publica¬ tions discuss various factors such as lunar synchrony which may effect the length of the human menstrual cycle. See "The Menstrual Cycle" by Rudolph V. Vollman, M.D., Vol. 7, Major Problems in Obstetrics and Gynecology, edited by Emmanuel A. Freidman, M.D.; Treloar, "Variation of the Human Menstrual Cycle Through Reproductive Life", International Journal of Fertility. 12 (1); 77-126 (1967). More recently, Winnifred Berg Cutler has reported upon the relation- ship between the lunar and menstrual cycle phases. See "Lunar and Menstrual Phase Locking", by Winnifred Cutler, American Journal of Obstetrics and

Gynecology 137:834 (1980). In this study, reference is made to data showing the mean and median of sample menstrual cycle data to be 29.5 days, and notes the coincidence of this cycle length and the 29.5 day lunar cycle. Cutler has noted that 98% of the cycles

+ of 29.5 - 1 day in length are ovulatory, and has suggested that ovulation may frequently occur during the new moon part of the cycle. In addition to the factors discussed above, sexual behavior has often been discussed as a factor which contributes to the rhythm of the human cycle and subsequent fertility. Cutler has published extensive¬ ly on the possible inter-relationship between in- fertility and first coitus, sexual behavior frequency and menstrual cycle length, short hyperthermic phases and sporadic sexual behavior in woman, and relationships between estrogen level, hot flashes and sexual behavior in perimenopausal woman. See Cutler et al "Infertility and Age at First Coitus; A Possible Association", J. Biosoc. Sci. .11:425-432 (1979); Cutler et al, "Sexual Behavior Frequency and Menstrual Cycle Length in Mature Premenopausal Women", Psvchoneuroendochrinology 4.:297-309 (1979); Cutler et al, "Luteal Phase Defects: A Possible Relationship Between Short Hyperthermic Phase and Sporadic Sexual Behavior in Women", Hormones and Behavior .13.: 14-218 (1979) ; Cutler et al, "Sporadic Sexual Behavior and Menstrual Cycle Length in Women", Hormones and Behavior 14:163-172 (1980); Cutler et al, "The

Psychoneuroendocrinology of the Ovulatory Cycle of Woman, A Review", Psychoneuroendocrinology 5_:1980; and Cutler et al, "Relationships Between Estrogen Level, Hot Flashes and Sexual Behavior in Perimenopausal Women", Neuroendocrinology Letters 5:185 (1983).

In addition to the Vollman and Treloar studies discussed above, which clearly suggest that women whose cycles approach a 29 day span have the highest likelihood of a fertile cycle, Cutler et al have demonstrated that women who have regular weekly, heterosexual activity have menstrual cycles of about 29 days, whereas women who either have sporadic sexual activity or who are celibate tend to have a higher frequency of aberrant length cycles (25 or less or 34 or more days) . These studies suggest that luteal phase defects (shortened hyperthermic phases) and sporadic sexual activity in the luteal phase are associated in a population of infertility patients. Delayed age of first coitus is also associated with a higher frequency of subsequent infertility. Even more recently, Culter et al have evaluated whether self stimulation (masturbation) , coitus, and/or heterosexual genital stimulation by a man without coitus, could be differentiated in terms of their relative association to fertility. In this study presumptive fertility (P. fertility) was evalu¬ ated by the menstrual cycle length and by analysis of basal body temperature charts. It was found that weekly heterosexual behavior is consistently associat¬ ed with menstrual cycle lengths of 29.5 — 3 days and that self stimulation behavior does not show a similar association between weekly behavior and circa 29 day menstrual cycle lengths. Women who report regular weekly sex with men are found to have a greater incidence of presumptively fertile menstrual cycles than those who report sporadic sexual activity. In the Cutler et al study the chief problem associated with less frequent sexual activity is less a failure to ovulate but rather a short luteal phase. A fertile menstrual cycle requires both an ovulation and an adequately long luteal phase to ensure sufficient time and steroid output to prepare the endometrium for implantation. The Cutler et al study shows that sexually active women who have regular, stable patterns of sexual activity have significantly more fertile type cycles than women of the same age who have sporadic patterns of sexual activity. The hypothesis that normalization of the menstrual cycle is due to something more than genital stimulation, but requires the presence of a male partner, is con¬ sistent with the suggestions of McClintock, the data of Quadagno, and numerous studies and observations conducted with non-human primates and other mammals, some of which suggest the presence of a male or his odors are important in regulating cyclicity and/or fertility. The influence of mammals or their odors on the estrous cycles of other female conspecifics has been well-documented in recent studies employing infra-human mammals. See McClintock, M.K. 1983, supra.. McClintock, M.K. 1984, supra.. Izard, M.K. 1983, supra. „ and Izard M.K. and Vandenbergh, J.G.

1982, supra. For example, the estrous cycle of rats can be manipulated through the use of odors collected from females during specific phases of the estrous cycle. In several rodent species, exposure to the odors of urine or cage bedding of males can promote estrous or stimulate ovulation. In some infra- human primates, males can influence cycle lenth. Female baboons denied a mating showed significant¬ ly longer cycles and rhesus monkeys showed a summer amenorrhea two to four months after a male decrease in sexual potency.

Notwithstanding what is known on this topic, the problems of unwanted fertility and unwanted infer¬ tility remain among the most important of human concerns. Since a reasonable percentage of infertility may stem from conditions reflected in menstrual cycles of aberrant lengths, methods for adjusting the lengths of those menstrual cycles have a good likelihood of creating presumptively fertile 29.5 — 3 day cycles. Similarly, for those women choosing to avoid pregnancy, the provision of a physiologically compatible method for stimulating infertile-type cycles would be of utmost utility. Finally, improved methods for regulating the timing of the menstrual cycle could improve the predictability of the occurrence of the fertile period and/or men¬ struation and expand the range of therapeutic possibilities for addressing both fertility and infertility problems. Summary of the Invention The present invention provides a novel method for altering and/or regularizing the length of the menstrual cycle of a human female. Applicants have discovered that the axillary secretions of males, when exposed to the nasal region of females with a history of aberrant. cycle lengths (<26 or >33 days) tend to induce normal, presumptively fertile menstrual cycles having lengths of about 29.5 - 3 days. Applicants have demonstrated that, as compared to control^'s receiving only solvent (ethanol) applications, woman receiving male axillary extracts experienced more regular cycles, and, based on BBT data, more presumptively fertile cycles.

In accordance with the preferred embodiment of the present invention, the male donor axillary secretions should be exposed to the nasal region of the female for a duration and in amounts effective to significantly alter or regularize the expected cycle length of that female. Such exposure should preferably be accomplished through periodic applications of male donor extract of the axillary secretions of one or more males. The method is particularly useful in treating women exhibiting aberrant menstrual cycles which are either monophasic, or which have lengths 26 days or less or 33 days or more. Menstrual cycles of 26 days or less are often indicative of luteal phase defects which are associated with presumptively infertile cycles. Accordingly, the present method is particularly adapted for increasing the presumptive fertility of those cycles by exposing a female having a menstrual cycle exhibiting a luteal phase of less than 12 days to the subject male donor axillary secretions for a period of time sufficient to increase the length of the luteal phase of the menstrual cycle to at least 12, and preferably to about 14-16 days. Accordingly, the present invention provides a novel material for application to the human body comprising axillary secretions of at least one human male donor, which material may be provided in a kit for altering. the length of the expected menstrual cycle of a female. The subject kit should comprise a series of medicaments, each of which contains at least a portion of the axillary secretions of a human male donor, whereby the medicaments may be administered periodically to the nasal region of the female to thereby alter the expected length of the female's menstrual cycle.

Accordingly, a primary object of the present invention is a method for altering the length of the menstrual cycle of a human female and regularizing it towards a normal 29.5 — 3 day menstrual cycle length.

A further object of present invention is the provision of the method for altering or regulating the length of the menstrual cycle of a human female to produce a greater incidence of presumptively fertile menstrual cycles.

A further aim of the present invention is the provision of a novel method for treating human females exhibiting luteal phase defects.

A further aim of the present invention is the provision of a method for improving the fertility of a human female. Another aim of the present inven¬ tion is the provision of a kit for altering the length of the expected menstrual cycle of a human female. These and other objects of the present invention will become apparent from the following, more detailed description.

Brief Description of the Drawings

Figure 1 shows the first cycle length (la) and last cycle length (lb) of all subjects receiving male axillary extract stimulus as compared to subjects receiving only a placebo;

Figure 2 is a chart in which data for woman having weekly sexual activity was removed from the data set of Figure 1, which shows the effect of the male extract stimulus on cycle length; Figure 3 are gas chromatography, profiles in which the Flame Ionization Detector (FID) responses are presented in the vertical axis and time is presented in the horizontal axis. This figure illustrates odors which elute when the extracts are chromatographed using different liquid phases.

Figure 3A shows the chromatogram on a non- polar silica phase (CP SIL-8) .

Figure 3b shows the chromatogram on a polar phase (CP-57 WAX) . Description of the Preferred Embodiments

The present invention provides a novel method for altering or regulating the length of the menstrual cycle of a human female towards a 29.5 • 3 day menstrual cycle. The studies reported herein are believed to be the first systematically designed, prospectively conducted, double-blind re¬ 86/0

-19- search which demonstrate that male axillary secre¬ tions can influence female endocrine events.

In order to investigate potential mechanisms controlling the association between heterosexual activity and menstrual cycle length, and in light of the non-human literature suggesting that a chemical signal from males could be involved, it was investiga¬ ted whether an extract from the male axillary region would a sufficient stimulus to induce presumptively fertile type cycles.

Axillary secretions were collected from three male volunteers (age 41, 35 and 32) . Each was engaged in a heterosexual relationship and had large numbers of lipophiliσ diptheroids in his axillary region. See Labows, J.N. 1979, supra. and Leyden, J.J., McGinley, K.J., Hoelzle, E., Labows, J.N. and Kligman, A.M. 1981, supra. In addition during the three months in which they collected secretions they were required to (1) not use deodorant, deodorant soap ox perfumes in the axillary region and (2) wash once in the morning with Ivory soap. Secretions were collected on a 4 inch x 4 inch cotton pads which had previously been extracted, autoclaved, dried and wrapped in solvent extracted foil. See Petri, G. and Huggins, G.R., "Cyclic Changes in Volatile Acidic Metabolites of Human Vaginal Secretions and their Relation to Ovulation", J. Chem. Ecol. 2.: 361-376 (1975) . Each donor wore a pad in each axilla three times a week during a six to nine hour period which was most convenient for him. Each day after removal, the pads from any one donor were placed in an individual acid-σleaned-glass-jar and frozen at -60°C. until extraction. Secretions were collected three times a week from each donor for a 12 week period. Preparation of extract was conducted after all pads had been collected and_.frozen. Batches of glass jars were grouped consecutively according to the day received. Six to eight jars containing pads from all three individuals were used to prepare each batch of pooled extract. A total of 14 separate batches were prepared in this manner. All pads from each batch were placed in a glass column and allowed to soak in doubly distilled ethanol (15 ml/pad) for one hour. The ethanol was allowed to drain from the column through a PTFE (Teflon) stop cock and the pads were squeezed with a PTFE (Teflon) disk. Recovery approached 66% of the ethanol added to the pads_* Pre¬ sumably the remaining 34% remained within the pads. The ethanol extracted stimulus batches were then stored at -60°C. Subsequently, 15 women, varying in age from

19 to 22 and one aged 30 were enrolled in and completed the study. All met the following criteria: "Gynecological maturity" (as defined by Treloar et al as menstruating for at least 7 years) , nulliparous, unmarried, not currently (nor within the last months) using oral contraceptives nor an IUD, and a willing¬ ness to make daily entries of basal body temperature (BBT) and sexual behavior. Each potential subject who indicated her interest in participating was asked whether she thought she had a short cycle (<26 days) , a normal type cycle (29.5 -^'3 days) or a long cycle (>33 days) . Those who indicated they had an aberrant length cycle (25 or less, or 34 or more days) were included in this study; those who indicated normal type cycles (29.5 — 3 day) were diverted to the study -which is reported in connection with the afore¬ mentioned related patent application concerning the use of female essence to alter the .timing of the menstrual cycle. In addition, all subjects agreed to provide blood samples for steroid analysis during three days of one week in the luteal phase of the last menstrual cycle studied. A complete history and physical exam was performed on each subject. This screening process for possible pathologies which might influence the length of the menstrual cycle failed to find any. Each subject was provided with basal body temperature (BBT) charts, BBT thermometers and a calendar card to record sexual behavior. Instructions for their use were given by a technician who was blind to the purpose of the study. Subjects were unaware of the true nature of the stimuli and were told only that they were receiving a "natural fragrance" extracted into ethanol. Thus the study was double-blind. Subjects were randomly assigned to group A (axillary extracts/ethanol) or group B (blank/ethanol) . The average age of group A was 20 while the average age of the B group was 22. Application of the stimuli began in September, 1983, but because of individual variation in menstrual patterns any particular women could enter the study at any point within her cycle. Subjects came into the laboratory three times each week for 13.5 — 1 weeks for application of the stimuli (male extract or placebo) . Each morning the previously frozen stimuli were removed and allowed to warm to room temperature for 30 minutes. Individual 5 milliliter samples were removed via pipet and placed in separate vials for transport to the laboratory where the stimuli were applied. One half milliliter of the stimuli (axillary extract of placebo/ethanol) was pipeted onto a clean 4 inch x 4 inch cotton pad. The technician rubbed the contents of the pad on the upper lip of the subject and instructed her not to wash the area for at least 6 hours.

Figure 1 shows the first cycle length (1A) and last cycle length (IB) of all subjects receiving stimulus. There was significantly less variance in cycle length for women receiving male stimulus then for women receiving the placebo (first cycle F_ - = 5.97, p less than .025; last cycle F_{Q 6} = 8.09, p less than 0.025). There was no over all difference in mean cycle length although a trend for this was evident by the last cycle (t = 1.45, p less than 10).

Because weekly heterosexual behavior and 29 day cycles are associated the data for those women who were having weekly sexual activity with men were removed from the data set; the data was then reanalyzed. See Cutler et al. (1980) supra. and Cutler et al: (1979) supra. The results are set forth in Figure 2. Again, the variances were significantly different (first cycle F_g _ = 11.56, p less than .01; last cycle F_{g 5} = 20.61, p less than .01) and although the mean cycle lengths appear to be different, the difference failed to achieve acceptable statistical significance t = 1.645, 0.05 p less than .10. Table 1 shows the incidence of aberrant cycles (<26 or >33 days) to vary as a function of treatment in all women (left) and when controlled for sexual activity (right) . Significance was not achieved until the last cycle. In studying either all the subjects or only those subjects who were not having.weekly sexual activity, exposure to male axillary secretions yielded a greater proportion of "normal" (29.5 - 3 day) cycle lengths than exposure to the placebo.

TABLE 1

INCIDENCE OF ABERRANT CYCLES VARIES AS A FUNCTION OF

TREATMENT AND SEXUAL ACTIVITY

All Subjects Less than Weekly Subjects

First Cycle

Treatment Treatment

Extract Blank Extract Blank

Aberrant 5 7 Aberrant 4 5 cycle cycle

Normal 2 2 Normal 2 1 cycle cycle ,

Z = .2936 Z = .688 n.s. n.s.

Last Cycle

Treatment Treatment

Extract Blank Extract Blank

Aberrant 2 6 Aberrant 2 5 cycle cycle

Normal 5 3 Normal 4 1 cycle . cycle

Z - 1.512 Z = 1.754 p .066 p .04

The percentage of women exhibiting a normal length luteal phase (at least 12 hyperthermic days) subsequent to receiving the male extract or placebo stimulus has also been determined from basal body temperature charts. Analysis of basal body temperature charts was performed along criteria pre¬ viously reported. See Cutler et al. (1980) supra. Among all of the women, the sexually active, or the sexually inactive, a higher percentage of male extract recipients than placebo recipients showed a normal length luteal phase basal body temperature graph (p less than .0625). See Cutler et al. (1979) supra. The consistency of the results lends credence to them. Levels of progesterone (3 samples per subject) supported the basal body temperature graph scoring method. In each case where the scorer recorded a presumptively fertile cycle, as well as with presumptive defects in the BBT, the progesterone values were within expected ranges for the days of the cycle assayed. There were no apparent differences in testosterone level, or estradiol level among women who received different treatments or who showed different patterns of sexual activity in this study.

The present invention thus provides a novel method for altering or regulating (normalizing) the length of the menstrual cycle of a human female towards 29.5 — 3 day menstrual cycles, comprising the step of exposing the. nasal region of said female to at least a portion of the axillary secretions of the male, said exposure continuing for a duration and being in amounts effective to significantly alter or regulate the expected cycle length of that female. While it is presently preferred to use extracts of male axillary secretions as said portions, it is theorized that the effective pheromonal component of the axillary secretions may be one or more of the androgen steroids: androstenol, androstenone, dehydroepiandrosterone, androstadienol, andro- standienone dehydroepiandrosterone sulfate (DHAS) , androsterone sulfate (AS) , and natural derivatives thereof. It is further anticipated that the burnt heavy axillary-like odor component which elutes at about 50 minutes as shown on the FID response curve of Figure 3B may also exhibit pheromonal activity. in any event, the portions of the male axillary secretions should be applied periodically for at least two days during each menstrual cycle. Preferably these secretions should be applied three times each week. When application throughout the course of the menstrual cycle is not feasible, it is presently preferred to apply the axillary extract at least during the follicular phase, and preferably also during the periovulatory phase, of the menstrual cycle. As shown from the data collected over the course of several menstrual cycles, application over at least several menstrual cycles is preferred, at least when the male axillary secretions are applied with the frequency and in the concentrations used in this study.

Good results may be obtained when the axillary secretions employed comprise secretions collected from more than one male donor. As mentioned above, it is preferred that such secretion be collected periodically from each male donor and be applied to the upper lip of the female, preferably on a daily basis. When the subject female is being treated for a short menstrual cycle, one in which the luteal phase is 11 days or less, application of the male axillary secretion should continue until luteal phases of at least 12 days are established. A similar regimen should be followed for aberrantly long menstrual cycles, and/or when human females are being treated which exhibit monophasic menstrual cycles as determined from basal body temperature graphs.

The present invention may be practiced by providing a kit for altering the length of the expected menstrual cycle of a human female, comprising a series of medicaments, each of which includes at least a portion of the axillary secretions of a human male donor, whereby the medicaments may be administer- ed periodically to the nasal region of the female to thereby alter the expected length of that female's menstrual cycle. A material is thus provided for application to the human body comprising the axillary secretions of at least one, and preferably a plural¬ ity, of human male donors. As mentioned above, the possible role that odors play in human reproductive biology has received considerable attention particularly with respect to the extent to which menstrual synchrony may occur in females who live together. Discussions of male effect on females have ofte centered on the possibility that odor acts to attract, or sexually arouse the male. The data presented here indicate that prolonged exposure to one or more constituents from the male axillae alters the female endocrine system. Since both the experimental and the control subject in this study spend a considerable portion of their day in a heterosexual environment, the causative factor in male axillary extract is likely to have a low vapor pressure at body temperature (or none at all) and critical concentrations may only be transferred during intimate contact. While the experimental protocol does not permit definitive determination as to whether the effect is mediated by olfactory stimuli or if the constituents causing the effect are absorbed through the skin, it is nonetheless clear that male extract applied to the nasal region of the treated female definitely effects the female menstrual pattern. These reasons suggest that the active agents in male axillary secretion may be the burnt heavy axillary- like component and/or the steroids mentioned above. As shown in Figure 3 when using a non-polar silicon phase two main odor-containing areas are seen: one near the solvent front and the second where the volatile steroids (androstenol and androsterone) and pyrolysis products from AS and DHAS elute. The area near the solvent front on the silicon phase is retained longer on the polar CP-57 WAX and elutes with a very marked axillary/burnt odor at approximately 50 minutes into the course of the chromatogram. The steroids do not elute on this column until well after 70-80 minutes after injection. Many of the short-chain aliphatic acids elute within the first 30 minutes and give sharp, acid-like odors.

Compounds giving rise to the odors eluting from the polar CP-WAX column at approximately 50 minutes may be of particular interest because mass spectrometry of this sample shows no distinct mass spectra in the region where this intense odor is found. Consequently, highly odiferous material exists but its concentration must be below the detection limit of our instrument (at full scan this is approximately 250-500 picograms) .

As seen from the above, novel methods for improving the presumptive fertility .of human females are disclosed based upon the unexpected discovery that males can exert a "primer" pheromone effect on the human menstrual cycle.

Claims

C L A I M S

1. A method for altering or regulating the length of the menstrual cycle of a human female towards 29.5 + 3 day menstrual cycle, comprising the step of exposing the nasal region of said female to at least a portion of the axillary secretions of a human male, said exposure con¬ tinuing for a duration and being in amounts effective to significantly alter the expected cycle length of that female.

2. A method for improving the presumptive -fertility of a human female comprising the step of exposing the nasal region of said female to at least a portion of the axillary secretions of at least one male donor.

3. The method of claim 1, wherein said portion is selected to comprise at least one compound selected from the group consisting of androstenol, androstenone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstadienol, androstadienone, androsterone sulfate, and natural derivatives thereof.

4. The method of claim 1, 2 or 3, wherein said portion is an extract of male axillary secretions.

5. The method of claim 1, 2 or 3, wherein said portion of said axillary secretions include the component of said secretions exibiting a burnt heavy axillary-like odor which elutes at about 50 minutes as shown in the FID response curve of Figure 3B.

6 _K The method of claim 1, 2 or 3, wherein said portion is applied periodically for at least two days during each menstrual cycle.

7. The method of claim 7, wherein said portion is applied periodically over the course of at least several menstrual cycles.

8. The method of claim 6, wherein said application is effected at least 4 separate days of said cycles and said applications are spaced apart on days throughout at least the follicular and periovulatory phases of each cycle.

9. The method of claim 8, wherein said times include at least times during each of cycle day periods 1-6, 7-12, 13-18 and 19-menses (wherein cycle day 1 is the first day of menstruation) .

10. The method of claim 1, 2 or 3, wherein said males have significant populations of lipophilic diptheroids in their axillary region.

11. The method of claim 1, 2 or 3, wherein said secretions comprise secretions collected from more than one male.

12. The method of claim 1, 2 or 3, wherein said female has an expected menstrual cycle exhibiting a luteal phase of less than 12 days and wherein said exposing is continued until the luteal phase of the menstrual cycle of said female has. lengthened to at least 12 days.

13. A kit for altering the- length of the expected menstrual cycle of a human female, comprising a series of medicaments each of which comprises at least a portion of the axillary secretions of a human male donor whereby said medicaments may be administered periodically to the nasal region of a female to thereby alter the expected length of that female'smenstrual cycle

14. A material for application to the human body comprising an extract of axillary secretions of at least one human male donor.

15. The material of claim 14, comprising the secretions of a plurality of said donors.

16. Use of an extract of axillary secretions of the human male to alter or regulate the length of menstrual . ^"cycle of a human female or improve the presumptive fertility of a human female.

17. Use of an axillary secretion of the human male to prepare a composition for altering or regulating the length of the menstrual cycle of a human female or for im¬ provingpresumptive fertility of a human female.