DIRECT BASOPHIL COUNT FOR TIMING OVULATION

Transcription

1 FERTLTY AND STERLTY Copyright 1974 The American Fertility Society Vbl. 25. No.8, August 1974 Printed in U.S.A DRECT BASOPHL COUNT FOR TMNG OVULATON LSE LOTTE METTLER, M.D., AND DARUS SHRWAN, M.D. Timing of ovulation still represents a major diagnostic problem in gynecology, especially in the treatment of sterility. From the wide spectrum of measurements available, only the preovulatory peak concentrations of estrogen, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and the postovulatory increased concentration of progesterone are reliable indices of ovulation. Their exact determination demands considerable expense for apparatus and technical assistance. n a study of hematologic changes during the menstrual cycle we found the Lasophil count of blood to be an additional aid for timing ovulation. This report describes our observations. MATERALS AND METHODS Each of 130 healthy women, 20 to 35 years of age, had a thorough hematologic study. Venous blood samples were collected ten times per menstrual cycle, always at the same time of day and no sooner than 4 hours after food intake. Ninety women had normal ovulatory cycles of approximately 28 days; 20 had anovulatory cycles and, in an additional 20 women who had been anovulatory, ovulation was induced by treatment with gonadotropins; Ovulation was determined by all hitherto available criteria: basal body Received July 25, Department of Obstetrics and Gynecology, University of Kiel, Germany temperature, cervical mucus (Spinnbarkeit, arborization, and the burning phenomenon), urinary ascorbic acid concentration, and radioimmunoassay of LH (LER-907), FSH, and progesterone (with antibody against progesterone-6o: bovine serum albumin), as well as by colorimetric measurement of total estrogens in the urine. The relative number of basophils was determined by differential leukocyte counts, counting a total of 5,000 leukocytes for each determination. The absolute basophil count was computed indirectly from the total leukocyte count and from the corresponding relative values. The basophils were also counted directly in Fuchs-Rosenthal hemocytometers. According to the method of Cooper and Cruickshank, 2 20 fll of fresh venous blood was mixed with 80 fll of 0.1% disodium ethylenediamine tetroacetic acid (EDTA) in saline, and then 100 fll of a dye solution was added. The latter consisted of equal volumes of 0.5% cetylpyridinium chloride and 0.8% toluidine blue (Merck) in 5% aqueous solution of aluminium sulfate and distilled water. A sample of the final mixture was pipetted into a Fuchs Rosenthal hemocytometer filling up the chamber by capillary action. The leukocytes were allowed to settle in the ruled area for at least 3 minutes. To avoid an error in concentration caused by 718 ~ ~...

2 Vol. 25, No.8 DRECT BASOPHL COUNT 719 ) evaporation, the chamber was kept in a moist atmosphere. The cells were counted under a lox or 25x objective of the microscope. Blood basophils were easily detected by the characteristic purple-red metachromasia of their cytoplasmic granules. Although the cetylpyridinium chloride solution destroys and eliminates the red blood cells and platelets, it also selectively stabilizes the glycosaminoglycans in the basophil gran~ ules. To these polyanions in the granules, toluidine blue, a cationic dye, strongly binds and produces the selective purplered metachromasia. Statistical treatment. The two-dimensional analysis of variance was applied to determine whether the results were significant (F>Fa). Using the Student's t test, the smallest total difference (Sx' - Sx") necessary for significance comparing the ovulatory basophil count to all the other nine basophil counts per cycle, was computed. number 01 patl.n" n 10l8.0 t. _. 27.' 12.0 _t batophll collnt FG. 1. Frequency distribution of blood basophil counts using the direct counting method for anunselected population of women of reproducthre age (n == 1,028). absolutrbmp.il count ptr mm' - indirect absolute basophil count direct absolute basophil count relati"bl$ iltountih% _ relative basophil count P<~05 10 a, J. 1. 't..4 ';L; '~ 1.~ 6~~ '., \- days Ovulation FG. 2. Comparison of blood basophil counts during the menstrual cycle measured by three methods: ndirect absolute basophil count, direct absolute basophil count, and relative basophil count. All points are the means of measurements made in 90 cases of spontanoous ovulation. Measurements significantly different (P<O.05) from the other days are indicated by the asterisk. RESULTS Blood basophil counts done previously using the direct method for more than 1,000 blood samples from women between 20 and 30 years of age revealed an approximately symmetrical frequency distribution that approached a, Gaussian curve (Fig. 1). The standard deviation of the direct counting method was +3.0%; the standard deviation of the basophil counts was %. Spontaneous ovulatory cycles. n cases of spontaneous ovulation, the basophil count by both methods remained rather constant during the first part of the cycle (Fig. 2). No statistically significant differences were found within this part of the cycle. At the time of ovulation a swift and statistically significant decrease in the number of basophils was measured. The number then increased during the luteal phase until it equaled the value measured initially. The difference, which was statistically significant, between the mean basophil counts for the 10 selected days of the cycle is expressed by F>FaO.05 [F = ; FaO.05 (f1.:..:.. 9, f2 = 801) = 1.89]. The smallest total difference

3 720 METTLER A:f\fD SHRWAN August 1974 blood basophil count per mm : total oestrogens JUg/ die 40-: : 1* ", *' *' LH ng /ml -800, ~ ",......,.. *p < 0.05 e :,, - ' 10-: \ ' " --,--,, "",' ' days FG. 3. Mean concentrations of basophils, total urinary estrogens, and serum. LH in 90 cases of spontaneous ovulation..... necessary for statistical significance, (Sx' - SX") 0.05 = 184, is present at all nine points of the cycle when compared to the time of ovulation. The significant decrease in number of basophils at midcycle coincided with the peaks in serum concentration of LH and urinary concentration of estrogens, which inqicated ovulation (Fig. 3). Ovulation was further defined by the increase in serum progesterone level, which followed the midcycle decrease of basophils (Fig. 4). Anovulatory cycles. n contrast to our observations in cases of spontaneous ovulation, no statistically significant changes in the basophil count were measured during the whole course of the menstrual cycle in 20 cases of anovulation (Fig. 5). Although the only evidence of ovulation that Figure 5 shows is the rise in the mean basal body temperature, its occurrence in all cases was confirmed by the other measurements mentioned previously. nduced ovulation. n 20 cases in which ovulation was induced by treatment with human menopausal and human chorionic gonadotropins, the basophil count decreased on the day of ovulation, as it did in cases of spontaneous ovulation. Figure 6 shows the mean number of basophils during the five days prior to and the five days after ovulation as compared to the mean levels of serum progesterone. Of the various measurements that were made to determine ovulation, only serum progesterone concentration is presented. ts rise indicated that ovulation had occurred, thereby placing it at the time of the midcycle decrease in basophils. The basophil count on the day of ovulation was significantly different from that on all other days, except for the fourth

4 )" Vol. 25, No.8 DRECT BASOPHL COUNT 721 serum progesterone pg/ml blood basophil count per mm 'Y 1000 *p < 0, ov days FG. 4. Mean concentrations and standard deviations of basophils and serum progesterone in 90 cases of spontaneous ovulation direct basophi l count per mm 3 -- :!: Sx anovulatory cycles - i± Sx ovulatory cycles B T , " ,.,..., Zo *p<0.05 r( FG. 5. Mean concentrations and standard deviations of basophils in 90 cases of spontaneous ovulation and 20 cases of anovulation shown with the mean basal body temperature during cycles with ovulation.

5 722 METTLER AND SHRWAN August 1974 ~ blood basophi count per mm 3 serum progesterone pg ", \ 1 ""..* /, /.~. '. ---:,: _-----_.--_.- *p < FG. 6. Mean concentration of basophils and serum progesterone in 20 cases of induced ovulation. The shaded area represents the day of ovulation defined by the subsequent increase in, serum progesterone concentration. day before ovulation, based on a twodimensional analysis of variance. DSCUSSON Blood basophils like other granulocytes of the peripheral blood are derived from promyelocytes in the bone marrow,1 The water-soluble granules in the cytoplasm of basophils contain considerable amounts of glycosaminoglycans and histamine. The extreme scarcity of basophils in peripheral blood (0.4% of total leukocytes) and the high water solubility of their granules have been major technical problems preventing detailed examinations. Most of the quantitative studies on blood basophils performed so far are, therefore, certainly marred by inaccuracy. The introduction into hematology of substances like cetylpyridinium-chloride 2 or 5-aminoacridine hydrochloride, 3 which precipitate acidic glycosaminoglycans, has eliminated this gap in technology. Boseila 4 observed a decrease in basophil count in female rabbits 12 hours after copulation; Thonnard-N eumann 5 proposed that blood basophils are influenced by estrogen and progesterone. The results of our study show a clear-cut dependency of the blood basophil count upon the special hormonal situation that characterizes ovulation. Both in cases of spontaneous (Fig. 3) and induced (Fig. 6) ovulation, a significant decrease in basophil count occurred on the day of ovulation. This phenomenon was not observed in cases of anovulation (Fig. 5). However, in our small group with induced ovulatory cycles, a transient but significant fall ih basophil count was measured on the fourth day before ovulation (Fig. 6). Since this early decrease in basophil count could only be detected in HMG/HCG induced ovulatory cycles, we are inclined to relate this finding to an early influence of estrogen.

6 Vol. 25, No.8 DRECT BASOPHL COUNT 723 As a routine diagnostic measurement, the basophil count has been used successfully in our laboratory as an additional tool for the detection of ovulation. The simplicity of counting basophils allows its application as a routine method even in small sterility clinics that do not have hormone assays available. As true of evaluation of cervical mucus and measurement of ascorbic acid, this measurement represents a far more reliable index for timing ovulation than measurement of basal body temperature, which is generally known to be inaccurate. Ovulation time can only be defined reliably if the blood basophil counts are made during a few consecutive cycles so the individual normal levels of blood basophis and the expected decrease in these levels are available for comparison. Because a basophil count takes less than 10 minutes, it can easily be completed and evaluated during a consultation. At present no satisfactory explanation of the mechanisms involved in the decreased basophil count associated with ovulation can be given. A likely hypothesis is that the basophils rapidly migrate into the ovarian tissue during ovulation and are there associated with rupture of the follicle. SUMMARY The subjects were 130 women of reproductive age who had a thorough hemotologic analysis ten times per cycle. The blood basophil count decreased significantly on the day of ovulation, which was determined by all available criteria including radioimmunoassays of steroid hormones and gonadotropins. Cases of nonqvulatory cycles did not show this decrease in basophil count. nduced ovulation in previously anovulatory subjects also was associated with a decrease in basophils. These findings indicate that blood basophil counts are a reliable criterion for timing ovulation. Because a direct basophil count can easily be performed within 10 minutes without major expense for equipment or technical assistance, it can probably be done during consultation even in small sterility clinics. REFERENCES 1. Parwaresch MR, Leder LD, Dannenberg KEG: On the origin of human basophilic granulocytes. Acta Haematol (Basel) 45:273, Cooper K, Cruickshank CND: mproved method for direct counting of basophil leucocytes. J Clin Patho119:402, Parwaresch MR: Der basophile Granulozyt. Habilitationsschrift, Kiel, Boseila AWA: Variation in the blood basophil count induced by sexual stimulation in the rabbit. Acta Endocrinol (Kbh) 30:477, Thonnard-Neumann E: The influence of hormones on the basophilic leucocytes. Acta Haematol (Basel) 25:261,1961