METHOD USING POOLED FEMALE AXILLARY ESSENCE FROM MULTIPLE DONORS TO ALTER MENSTRUAL CYCLE TIMING

Abstract: A novel method of altering the length of the menstrual cycle of a human female. The method includes the step of exposing the nasal region of the female to at least a portion of the pooled axillary secretions of a plurality of woman donors. These secretions are selected according to the exhibited cycle behavior of the donor. Such behaviors include cycle length, BBT behavior and/or lunar synchrony. The subject secretions are pooled in phase sequence, although different donor days may be combined to provide ''combined donor day'' stimuli which are effective to alter menstrual cycle timing.

Description: METHOD USING POOLED FEMALE AXILLARY ESSENCE FROM MULTIPLE DONORS TO ALTER MENSTRUAL CYCLE TIMING 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 hystologiσal 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 biphasiσ 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-immunoassay 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, Gynaecoloσia. 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", Physiol. 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". In: 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. Renrod. Fert. 66:189-196 (1982). Notwithstanding these and other reports of elaborate phero onal 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, 219:244-245 (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 -6- 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.. 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", Psychoneuroendocrinolocry_ 1:245-252 (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. Graham 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", Psvchoneuroendocrinol. :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- ent 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 apoσrine 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 microflora 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. 85_:80-90; an d Brooksbank, B.W.L., Brown, R. and Gustafsson, J.A., "The Detection of 5 -androst-lβ- en-3-ol in Human Male Axillary Sweat", Experientia 3j3:864-865 (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, Maior 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 Gvnecologv 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 Pre enopausal Women", Psychoneuroendochrinology 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 .13:214-218 (1979) ; ' Cutler et al, "Sporadic Sexual Behavior and Menstrual Cycle Length in Women", Hormones and Behavior 11:163-172 (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 Voll an 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 σyclicity and/or fertility. The influence of mammals or their odors on the estrous cycles of other female cpnspecifiσs 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 It has been found that the timing of the menstrual cycle of a human female may be altered by exposing the nasal region of that female to at least a portion of the pooled axillary secretions of a plurality of woman donors. These secretion portions are preferably selected according to the exhibited cycle behavior of the donors. When exposed to such pooled secretion portions in significant amounts and durations, the timing of the subject female's menstrual cycle may be rapidly altered. In accordance with the preferred embodiment of the present invention, secretions are collected from each woman donor periodically throughout her menstrual cycle. Each secretion sample is categorized according to the timing of its collection relative to the donor's cycle, and is pooled-with other samples from the same day or time period of other donors' cycles. For example, collections from days 1, 2 and 3 of each donor's menstrual cycle may be pooled to form a "Combined Donor Day 1" sample. Samples from donor days 4, 5 and 6 may be pooled as "Combined Donor Day 4", and so on, to yield ten pooled axillary secretion samples for application to the female to be treated. Unlike previously reported studies wherein a single relatively unique "driver" female was the sole donor of axillary materials, the present invention utilizes pooled axillary secretions from a number of women meeting various general criteria, the principal one of which is an exhibited regular 29.5 + 3 day menstrual cycle. Accordingly, reliance on sometimes anecdotal evidence that a given woman is a "driver" is not necessary to accomplish the objectives of the present invention. Moreover, the technique of the present invention apparently produces a more rapid alteration in the timing of the subject's menstrual cycle than other techniques relying on secretions from a single "driver" female. This is particularly remarkable in view of the fact that the secretions are pooled not only from different donors, but also from different days within each donor's cycle. In accordance with the preferred method, exposure of the nasal region of the female to the pooled axillary secretions should occur periodically, as for example, by applying an extract of those axillary secretions to the skin region between the nose and the upper lip of the female, such that the female is caused to smell said secretions. It is presently preferred that said portion contain at least the hormone containing portion of those axillary secretons, particularly the dehydroxyepiandrosterone sulfate and androstenol portions of those secretions. The exposure step should comprise exposing the portions to said female at periodic times, on at least on four, preferably on ten separate days, during each menstrual cycle. These exposures should be spaced apart on days throughout at least the follicular and periovulatory phases of each cycle at a periodicity which generally corresponds to the periodicity of the pooled stimuli. Exposure times will preferably include times during each of day periods 1-6, 7-12, 13-18 and 19-menses, and more particularly on about every third day of the menstrual cycle. It is presently hypothesized that the length and timing of the human menstrual cycle is subject to the influence of pheromones which have as their primary source the axillae. As with certain other mammalian species which have been studied, it is believed that the sharp rise in certain axillary secretion components, particularly the androstenol and dehydroxyepiandrosterone sulfate components, which peak in concentration during the human follicular phase, tend to shorten the menstrual cycle. By analogy to other mammalian species, the exposure of females to periovulatory secretions is expected to substantially lengthen the menstrual cycle, and therefore is expected to be of significant importance in treating luteal phase defects. Accordingly, a primary object of the present invention is the provision of a method of altering the timing of the menstrual cycle of a human female. Another object of present invention is the provision of a kit for altering that timing. These and other objects of the present invention will become apparent from the following, more detailed description. Description of the Preferred Embodiments The present invention provides a novel method for altering the length of the menstrual cycle of a human female. In accordance with this method, the nasal region of that female is exposed to at least a portion of the pooled axillary secretions of a plurality of woman donors, said secretion portion being selected principally according to the exhibited cycle behaviors of each of said donor. The exposure of such portions to the nasal region should continue in amounts and for a duration effective to significantly alter the cycle timing of that female. In order to demonstrate the subject method, a double-blind study was designed using a combine axillary extract from one group of female donors. When this extract was applied to a separate group of females, the recipients' menstrual cycle approached menstrual synchrony with the donor females' menstrual cycle. Axillary secretions were collected from five female donors recruited from among co-workers and members of the community. All had the following characteristics: they were engaged in a heterosexual relationship; they had large numbers of lipophiliσ diptheroids in their axillary region; and for the duration of the experiment they did not use deodorant, deodorant soaps or perfumes in the axillary region; and they washed- each morning only with Ivory soap. Axillary secretions comprise secretions from the apocrine, sebaceous and eccrine glands and are excellent substrates for permanent populations of lipophilic diptheroids. It is these lipophilic diptheroids that are believed to generate a variety of odoriferous materials including volatile steroids which have been associated with the unique body odor produced by the axillae. In addition, lipid secretions from sebaceous glands also serve as an odor fixative and the presence of body hair assists in odor dispersal as well as providing for an increased surface area for odor production. Secretions were collected on 4 inch x 4 inch cotton pads which had been previously extracted, autoclaved, dried and wrapped in solvent extracted aluminum foil. Each donor wore a pad in each axillae three times a week during a convenient 6 to 9 hour period. After removal, pads were immediately frozen in glass jars which had previously been acid/solvent cleaned and stored at -60°C. until extraction, approximately 1 year later. Each donor collected secretions for three complete menstrual cycles. However, the axillary pads included in the preparation of the stimulus came from only five of the 15 donated cycles. These cycles were selected because they met the following criteria: each was 29.5 + 2 days in length; the basal body temperature charts were clearly biphasiσ and presumably ovulatory with post-ovulatory thermal rises of 12 or more days; and menstruation occurred within seven days of a full moon. To prepare this stimuli, pads were grouped in three day segments: e.g., all pads from cycle days 1-3 were combined, called "combined donor day one" (the first day of menstruation equals day one of the menstrual cycle) . All pads in days 4-6 were combined and called "combined donor day 4"; all pads in days 7-9 were combined to form "combined donor day 7", and so on. In this fashion, ten separate extracts were prepared, each containing odors from different portions of the menstrual cycle to form the "donor cycle" of combined donor days 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28. All days from each 3 day group were placed in a glass column and allowed to soak in doubly distilled ethanol for one hour at room temperature. 15 Milliliters of ethanol were used for each pad in the column. After one hour the ethanol extracted materials were allowed to run out the bottom of the column through a PTFE (Teflon) stop cock as the pads were squeezed with a PTFE (Teflon) disk. Approximately two thirds of all ethanol put on the pads was recovered. The ethanol extracted stimuli were subsequently frozen at -60°C. until needed. Female subjects who were to receive the axillary extracts were recruited from amongst members of the university community. The nineteen subjects who completed the study ranged in age from 19 to 32. All met the following criteria: "gynecological maturity" (as defined by Treloar et al as menstruating for at least 7 years) , nulliparous, not currently (nor within the last 3 months) using oral contraceptives nor an IUD, and a willingness to make daily entries of basal body temperature (BBT) and sexual behavior and menstrual occurrence. Each potential subject who met the criteria and indicated her interest in participating was asked if her menstrual cycle was normal (29.5 + 3) or aberrant in length (i.e. <26 or >33 days) . Those who indicated a normal type cycle were included in this study; the aberrant cyclers were placed in a study relating to the effect of male axillary extract, which is reported in the above-identified related patent application. All subjects also agreed to provide blood samples for steroid analysis during 3 days of one week in the luteal phase of the last menstrual cycle studied. Each subject was provided with 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; they were told they were receiving "natural fragrance" extracted into ethanol. Thus, the study was double-blind. Subjects were randomly assigned to group A, female axillary extracts/ethanol or group B, blank/ethanol. The average age of the A group (n=10) receiving female extract was 25.5 years while the average age of the B group (n=9) receiving blank/ethanol was 21.5 years. Because of individual variation in menstrual patterns a woman could enter the study at any point within her cycle. Upon entry, woman received female extract from "combined donor days 1". Sequential presentations of the remaining extracts were performed during subjects' subsequent visits to the laboratory (e.g., on her next visit donor day 4 is applied; on her next visit combined the donor day 7 extract and so on) . Subjects came into the laboratory three times a week for 11.5 _» 1.5 weeks and received an application of either the timed female extract or blank/ethanol in the following manner. Each morning the stimuli to be used that day were removed from the deep freezer and allowed to warm to room temperature for thirty minutes. Then individual 5 ml samples were removed via pipette and placed in separate vials for transport to the laboratory where the stimuli were applied. One half ml of the stimuli (axillary extract or blank/ethanol) was pipeted on a clean 4" X 4" 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 six hours. From the data obtained the difference in menses onset in days (- SEM) was determined between the menstrual bleeding on said of group A or B and the "menses" (combined donor day 1) of the "donor cycle" across the three months of the study. Table 1 lists the difference in days of menstrual bleeding onset for each individual subject and the application of the combined donor day 1 extract. TABLE 1 NUMBER OF DAYS BY WHICH A GIVEN CYCLE ONSET VARIED FROM DONOR MENSES SUBJECTS RECEIVING FEMALE AXILLARY EXTRACT Subiect Menses 1 Menses 2 Menses 3 1 12 6 11 2 11 5 7 3 11 5 2 4 13 2 5 5 8 2 4 6 1 3 1 7 6 10 5 8 6 4 1 9 3 1 3 10 6 5 0 Avg 7.70 4 .30 3.90 SEM 1.27 0 .82 1.04 Subjects receiving ] Blank/Ethanol Subiect Menses 1 Menses 2 Menses 3 1 5 3 8 2 7 2 9 3 4 9 9 4 1 3 0 5 4 1 1 6 10 11 15 7 12 9 8 8 8 2 9 9 9 13 12 Mean 6.67 5.89 7.89 SEM 1.15 1.52 1.58 The mean difference in group A menses onset and the "donor day" menses (i.e., combined donor day 1) was 7.7 days. For group B this was 6.7 days, both near the expected difference of 7.38 (each potential subject who met the criteria and indicated her interest in participating indicated that her menstrual cycle was normal (29.5 + 3 days, thus making 7.38 days the expected difference) . After two more months of female extract application the difference in menses onset relative to the "donor cycle" was significantly reduced (by almost 50%) for group A to 3.9 days while no change was evident for group B. An analysis of variance with repeated measures showed a significant month X group interaction F(2,34) = 4.27; P less than 0.05. No other term was significant in the analysis. Post-hoc tests demonstrated that the experimental group showed a significant shift in menses onset during the periods monitored (F [2,18] = 6.03; p less than 0.01); the.control group showed no such effect (F [2 / 18] = 1.36; p = 0.28). Other post-hoc tests also revealed that the control and experimental groups differed only in the third menses period. These data demonstrate the constituents from the axillary region of a group of women can be frozen for a period of one year, then thawed and combined in pooled batches that can alter the menstrual cycles in another group of women. This occurs in the absence of social contact between the donors and recipients. The donor constituents do not have to be from either the same cycle or same female. Although experiments with rodents have demonstrated that air bourne odors can produce estrous synchrony, the design of this study does not permit conclusions about whether the synchrony effect is mediated by olfaction or absorption of the correct constituents. However, the subjects in this study worked and/or attended classes in a heterosexual environment. All were either engaged in or were "in-between" a heterosexual relationship and presumably had a close circle of female friends which could influence cyclicity. Despite this, the cyclicity of subjects receiving combined female extract was rapidly altered well within the three or four months discussed by McClintock, or the four to five months shown by Russell's subjects. The short time required to demonstrate synchrony with the donor cycle indicates that using a stimulant from a group of donors provides a stronger stimulus than using the stimulation from a single donor. Thus, in the strength and frequency used here, application of a combined female axillary extract can alter the menstrual cycle of normal cycling women. In view of the above, it is believed that axillary secretions constituents from women with an abnormally short (less than 11 day) luteal phase can disrupt the cycle length of women with both previously documented normal cycle lengths and basal body temperature (BBT) rhythms. It is anticipated that the length of the BBT hyperthermic phase - the putative luteal phase - as well as the pre-thermal nadir (folliσular) phase may be varied using this technique. A shortening of the normal cycle length should occur in response to the treatment. These treatments are analogous to the odor-mediating phenomenon that have been shown in the rat. Follicular phase secretions are expected to cause a significant decrease in cycle length relative. . to the subjects receiving any of the other extracts. Periovulatory phase extracts are expected to lengthen the cycle of subjects receiving them while extracts from the luteal phase may have no effect on cycle length. The basic working hypothesis is that active ingredient(s) of axillary secretions are able to accelerate and/or retard the rate of hormone secretion/activity and thus change the length of one or both of the major phases of the menstrual cycle. One of the three donors phase specific samples (follicular, periovulatory, midluteal) may indeed be the critical donation, thereby indicating that application of the entire sequence may be unnecessary. It is presently anticipated that gas chro atograph- able constituents from each of the 10 donor days may be isolated for presentation to the subjects, and that application of only the critical components may be necessary in order to adjust the length of the treated females menstrual cycle. It is further anticipated that kits for altering the length of the expected menstrual cycle of a female may ultimately be provided comprising a series of medicaments each of which comprises at least a portion of the axillary^secretions of a woman donor who exhibits a different cycle length from that of the female to be treated. This series of secretions will have been collected at periodic times during the donor's menstrual cycle, whereby the medicaments may be administered periodically to the nasal region of a female to thereby alter the expected length of that female's menstrual cycle. When such a kit is to be used for birth control purposes, axillary secretions of at least one woman donor who exhibits menstrual cycles which are monophasic and/or whose menstrual cycles have luteal phases of less than 11 days are preferred. It is nonetheless preferred to provide such secretions from a plurality of donors. As seen from the above, the present invention provides a novel technique for altering the timing of the menstrual cycle of a human female which includes the step of exposing the nasal region of that female to at least a portion of a pooled axillary secretions of a plurality of women donors whose secretions were collected and pooled according to the exhibited cycle behavior of those donors. When the exposure was in amounts for a sufficient duration, the timing of the menstrual cycle of the treated female was significantly altered.

Claims: C L A I M S 1. A method of altering the timing of the menstrual cycle of a human female comprising the step of exposing the nasal region of said female to at least a portion of the pooled axillary secretions of a plurality of woman donors, said secretions portion being selected according to the exhibited cycle behavior of said donors, said exposure continuing for a duration and being in amounts effective to significantly alter the timing of the menstrual cycle of that female. 2. A method of treating a human female exhibiting menstrual cycles of longer or shorter than 29.5 + 3 days to induce a menstrual cycle of 29.5 + 3 days, com¬ prising the step of exposing the nasal region of said female to at least a portion of the pooled axillary secretions of a plurality of woman donors exhibiting menstrual cycle lengths of about 29.5 + 3 days, in amounts and for a duration sufficient to adjust said female's cycle towards a 29.5 + 3 length. 3. A method for " improving the presumptive fertility of a human female comprising the step of a exposing the nasal region of said female to at least a portion of the axillary secretions of at least one woman donor exhibiting a menstrual cycle length of 29.5 + 3 days. 4. The method of claim 1, 2 or 3, wherein said secretion portion is selected according to the cycle length of said donors. 5. The method of claim 4, wherein said cycle length is 29.5 + 3 days. 6. The method of claim 1, 2 or 3, wherein said secretion portion is selected according to the BBT behavior of said cycle. 7. The method of claim 6, wherein said BBT behavior is biphasic. 8. The method of claim 1, 2 or 3, wherein said secretion portion is obtained from donors whose menstration occurs within one week of a full moon. 9. The aethod of claim 1, 2 or 3, wherein said secretions are pooled according to menstrual cycle phase. 10. The iTtethod of claim 9, wherein said secretions are pooled in §hase from different menstrual cycle days of said donors. 11. The method of claim 1, 2 or 3, wherein said portion is selected to comprise follicular phase axillary secretions. 12. The --aethod of claim 1, 2 or 3, wherein said por¬ tion is selected to comprise at least cycling concentrations of androstenol or dehydroepiandrosterone. 13. The method of claim 1, 2 or 3, wherein said portion is selected tar comprise -periovulatory phase axillary secretions. 14. The method of claim 1, wherein said portion of said secretions is collected periodically from each of said donors as a series of portions and said series are exposed in sequence with the phases of the cycle of said female. 15. The method of claim 14, wherein said sequence is synchronous with the expected cycle of said female, to thereby regulate the female's cycle. 16. The method of claim 14, wherein said phase sequence is asynchronous with the expected cycle of said female to thereby disrmt said female's expected cycle. 17. A kit for altering the timing of the expected menstrual cycle of a female, comprising a series of medicaments each of which comprises at least a portion of the pooled axillary secretions of a plurality of woman donors who exϋbit a selected cycle length, said series of secretions having been collected at periodic times dur¬ ing each of .said donor's menstrual cycle, whereby said medicaments nay be administered periodically to the nasal region of a fβnale to thereby alter the expected timing of that female's expected menstrual cycle. 18. A material for application to the human body comprising the pooled axillary secretions of at least a plurality woman donors who exhibit menstrual cycles which are biphasic and have lengths of 29.5 + 3 days. 19. The method of claim 2, wherein said periodic times are in at least 4 separate days of said cycle. 20. The method of claim 19, wherein said periodic times are spaced apart on days throughout at least the follicular and periovulatory phases of each cycle. 21. Use of an extract of an axillary secretion of a human female to regulate the menstrual cycle or improve the presumptive fertility of a human female. 22. Use of an axillary secretion of a human female for preparing a composition for regulating the menstrual cycle or to improve the presumptive fertility of a human female.

Inventors: CUTLER WINNIFRED B, LAWLEY HENRY J, PRETI GEORGE, CUTLER, WINNIFRED, B., LAWLEY, HENRY, J., PRETI, GEORGE
Applicants: MONELL CHEMICAL SENSES CENTRE, MONELL CHEMICAL SENSES CENTER
Application year: 1986
Main classification: A61K35
IPC codes: A61K31, A61K35