Characteristics of donor semen and cervical mucus at the time of conception

Transcription

1 FERTLTY AND STERLTY Copyright 1983 The American Fertility Society Printed in U.8A. Characteristics of donor semen and cervical mucus at the time of conception Ar Edvinsson, M.D. * Per Bergman, M.D.* Yvonne Steen, Ph.D.* Stig Nilsson, M.D. H Ostra sjukhuset and Sahlgrenska sjukhuset, Gothenburg, Sweden With freshly ejaculated semen from 15 donors, 1017 inseminations were performed in 247 women. One hundred conceptions were achieved. The degree of crystallization of the cervical mucus and the characteristics of the donor semen were examined at each insemination. One insemination only from one single ejaculate was performed in each cycle. This procedure made it possible to describe the quality of the cervical mucus and the sperm sample characteristics on each occasion of conception. The conception rate was significantly reduced (P < 0.001) when the degree of crystallization of the cervical mucus was impaired. The conception rate was low when the sperm density was less than 20 million/ml, the percentage of abnormal sperm was above 50, and the degree of motility was graded 2 or less. The in vitro capillary sperm penetration test gave reliable information on the progressive motility of the sperm, showing a good correlation between penetration ability and conception rate. Fertil Steril39:327, 1983 Evaluation of the male fertility potential is principally based on analyses of semen characteristics. The semen properties most often correlated with fertility are sperm density, morphologic characteristics, and motility. The ability of sperm to penetrate cervical mucus, the white of hen's egg, or synthetic migration media are not routinely used, although the capillary migration tests have been shown to give reliable information on fertility potential. l Received July 7, 1982; revised and accepted November 23, *Department of Obstetrics and Gynecology, Ostra sjukhuset. tdepartment of Urology, Sahlgrenska sjukhuset. *Reprint requests: Stig Nilsson, M.D., Associate Professor, Department of Urology, Sahlgrenska sjukhuset, S Gothenburg, Sweden. Several standards for evaluation of semen quality have been published. 2-7 The limits of fertile characteristics, however, cannot be based on examination of sperm samples from men with proven fertility requesting sterilization, because they could be past the child-producing period. n infertile marriages childlessness is due to the wife in approximately 40 of cases, and, therefore, such marriages include fertile men. n consequence, it has been difficult to define the minimum standards of fertile sperm characteristics from investigations of sperm samples from these groups of men. Furthermore, semen quality varies between ejaculates from the same individual, and definite conclusions on the fertility potential therefore can hardly be made from a single sperm sample, or even two. The object of this report is to assess the properties of the fresh donor semen sample causing con- Edvinsson et a. Semen and cervical mucus at conception 327

2 NO. OF PATENTS , r-! ~.----,...2.! YEARS Figure 1 Distribution within age groups at insemination of247 women. ception and to study to what extent the quality of the cervical mucus was related to the conception rate. MATERALS AND METHODS The series was composed of all women who had consented to artificial insemination by donor (AD) using fresh donor semen from April 1976 until August The mean age of the 247 subjects was 30 years (range, 21 to 40 years). The distribution within age groups is presented in Figure 1. On clinical examination all women had normal gynecologic finclings. Tubal passage was proven by hysterosalpingography or laparoscopy in most of the subjects. Women with evidence of bilateral tubal occlusion were excluded from the study. The indications for AD are listed in Table 1. Oligoasthenozoospermia was defined as a count of < 5 million! ml, and motility of < 30. All couples had been involuntarily childless for at least 2 years. A complete medical history and physical examination were performed for each husband by a urologist, and repeated semen samples were analyzed. On the basis of these examinations, it was considered almost impossible for some male subjects to impregnate their wives; others were included as subjects on the basis of hereditary diseases or Rh incompatibility. Semen of 15 physically and mentally healthy donors was used. No donor had a hereditary disease in his family, and all had a normal karyo- type. Tests were performed for venereal disease and blood typing before the donor was accepted. At least two semen samples were analyzed, and the minimum requirements were a sperm count of 40 million!ml with 50 motile sperm and normal morphologic characteristics in at least 50 of the spermatozoa. A total of 812 ejaculates was obtained. On 205 occasions the ejaculate was divided and used to inseminate two women. The semen samples were collected by masturbation into a plastic condom. nsemination was always performed within 1 hour after delivery of the ejaculate. From each sample a probe was analyzed concerning sperm density, morphologic characteristics, and motility. Sperm density was counted in a Buerker counting chamber (Scherf Company, Germany). The morphologic evaluation was based on the principles described by Eliasson. 8 The percentage of motile sperm and the degree of motility were determined from microscopy at room temperature. The percentage of motile sperm was calculated to the nearest 5 level. Forward progression was rated from 0 to 4, 4 describing a very high degree of progressive motility. Sperm penetration ability was determined by the Kremer in vitro penetration test,9 with our modification of using a certain fraction of hen's egg white as penetration medium. When a fresh egg is crushed in a dish, the egg white floats outward. One outer liquid part and one highly viscous inner part surrounding the yolk are easily identified. n the boundary between these two media there is a thin layer of egg white that can be used as a penetration medium. n our laboratory we have found a significantly positive correlation between sperm penetration ability in ovulatory cervical mucus and in this intermediate part of the egg white.lo After incubation for 1 hour at 37 C in a moist environment, the capillary was examined in a microscope at a magnification of x 100. Readings were made of the distance, in millimeters, penetrated by the main wa:ye of spermatozoa (approx- Table 1. ndications for AD ndication Severe oligoasthenozoospermia Azoospermia Autoimmunization to spermatozoa Severe hereditary disease Ejaculatory incompetence Rh incompatibility Total No Edvinsson et al. Semen and cervical mucus at conception Fertility and Sterility

3 Table 2. Number of nseminations, Conceptions, and Conception Ratio of Each Donor No. of No. of Conception Donor inseminations conceptions ratio A B C D E F G H J K L M N Total imately 25 sperm per visual field). This reading was called the penetration level. The appropriate time for insemination was determined in each woman by examination of several consecutive basal body temperature records. Females with irregular cycles were treated in an attempt to regulate ovulation. Before each insemination, a sample was drawn from the cervical mucus for examination. The crystallization degree of the cervical mucus was judged by the same experienced investigator and graded from 0 to One insemination only from a single ejaculate was performed in each cycle. Only intracervical inseminations were used; intrauterine inseminations were avoided. We performed inseminations by instilling the ejaculate in small doses for 30 minutes into the lower part of the cervical canal, using a syringe and a plastic tubing. When antisperm antibodies had been shown in the male seminal plasma, the couple was advised to avoid sexual intercourse 2 days prior to AD. This advice was given because the husband's seminal plasma could agglutinate or immobilize donor's spermatozoa. ll RESULTS After 1017 inseminations, 96 of the 247 women (39) conceived a total of 100 pregnancies. The average number of inseminations required for conception was Pregnancy wastage was 16 (15 abortions and 1 ectopic pregnancy, 2 spontaneous abortions occurring in 4 women). Of 84 pregnancies terminating in live births, there were 2 sets of twins. There were 50 boys and 36 girls. No significant difference in semen properties was found between sperm leading to abortion or live birth;. nor was any difference found in semen properties between samples leading to a male or a female child. The conception rate of each donor is presented in Table 2. t varied from 4.7 to 20.6 for 12 of the 15 donors. Two pregnancies were achieved after four inseminations using semen from one donor. No pregnancy occurred in 38 women following 40 inseminations using sperm delivered by 2 donors. The influence of the cervical mucus factor, judged from the degree of crystallization, is presented in Table 3. The quality of the mucus could not be analyzed in eight women because of blood contamination. A significantly reduced pregnancy rate was found when the degree of crystallization of the cervical mucus was less than (X 2 = 16.6, P < 0.001). Since the properties of the cervical mucus influenced the/conception rate, in the investigation of sperm fertility characteristics we have used only inseminations in women with the degree of crystallization of n this series of women 89 conceptions occurred following 722 inseminations. The average number of inseminations required for conception was 8.l. The relationship between sperm density and the conception rate is shown in Table 4. The conception rate was very low when the sperm density was less than 20 million/ml, but the number of sperm samples with such low density was small in this study. The relationship between the morphologic characteristics of sperm and the conception rate is shown in Table 5. The pregnancy rate was reduced when the percentage of abnormal sperm exceeded 50. No difference in the conception rate Table 3. Conception Rate According to Degree of Crystallization of Cervical Mucus Degree of No. of No. of Conception crystallization inseminations conceptions rate P < Total Edvinsson et al. Semen and cervical mucus at conception 329

4 Table 4. Relation Between Conception Rate and Sperm Density Following 722 nstances of AD Spenn No. of No. of Conception density inseminations conceptions rate 109/1." < was found between the groups containing less than 50 abnormal sperm. The conception rate was significantly improved when the percentage of motile sperm exceeded 40 (X 2 = 7.17, P < 0.01) (Table 6). The pregnancy outcome following 76 inseminations with sperm samples containing more than 50 motile sperm was very high (22.4), indicating motility to be of great importance in fertilizing capacity. When the degree of motility was graded from 0 to 4, the conception rate was low only on occasions when the degree of motility was 2 or less (Table 7). n fact, no pregnancy occurred following AD using sperm samples belonging to this category. The ability of spermatozoa to penetrate egg white in the capillary penetration test was examined in samples from 542 inseminations. The degree of crystallization of the female cervical mucus was grade in 369 of these samples. The conception rate in this group was over 13 when the penetration level was 12 mmlhour or more, 10.9 when the penetration level was 10 or 11 mmlhour, and 5.7 when the penetration level was less than 10 mm/hour (Table 8). Thus, a close correlation was found between the capillary migration test and the conception rate. DSCUSSON The pregnancy rate in this series of 247 women submitted to AD was 39. One reason for this relatively low pregnancy rate might be that only one AD was performed in each cycle. Another contributing factor is that during the investigation time, 69 women were inseminated three times only or less. The women had stable marriages with longlasting infertility with the present husband. The semen of their male partners was at repeated examinations found to be of a quality so poor that the possibility of a conception could almost be excluded. t is thus reasonable to believe that the semen used for the AD was responsible for the conception, and that the semen characteristics reflected the quality responsible for the conception. t must be emphasized, however, that the biologic situation at insemination is not identical to that of normal coition. All sperm donors were accepted on the basis of high semen quality, evaluated after investigations of repeated specimens. However, even semen from donors with proven high quality was sometimes rather poor, an observation verifying the results of previous studies,12, 13 emphasizing the importance of repeated semen analysis for the evaluation of the male fertility potential. The occurrence of multiple semen samples of rather poor quality from men who previously delivered multiple samples of high quality also stresses that the sperm quality of a male subject varies from timeto-time and that the quality of a man's sperm can be reduced without known reasons. The quality of the cervical mucus is of great importance for the occurrence of conception. Dur- ' ing the menstrual cycle the mucus undergoes cyclic changes also regulating the possibility of sperm penetration. Several physical properties can be used to describe the character of the cervical mucus. n this study we examined the degree of crystallization in a semiquantitative way, using the four degrees of 0 to + + +, the degree of reflecting cervical mucus of high quality at the time of ovulation. A conception rate of 3.8 among women with a degree of mucus crystallization less than shows that examination of the mucal ferning is a relevant parameter in judging the quality of the mucus and in determining the appropriate time for insemination. However, in spite of a degree of crystallization of + + or less, 11 conceptions occurred, indicating these inseminations to have been performed close to the time of ovulation. Conception in spite of a reduced Table 5. Number of Conceptions and Conception Rate According to Percentage of Abnormal Sperm Percentage of No. of in- No. of Conception abnonnal spenn seminations conceptions rate < " Edvinsson et a. Semen and cervical mucus at conception Fertility and Sterility

5 Table 6. Conception Rate According to Percentage of Motile Sperm Motile No. of No. of Conception sperm inseminations conceptions rate ;; ;;: P < 0.01 degree of crystallization in these women might reflect that the analyzed part of the mucus might not be representative of the entire secretion and that the mucus in the upper part of the cervix was of a better quality. Poor quality of the mucus at the time of examination could also reflect an unknown defect or disease in the cervix. The application of the ejaculate in the cervix could also facilitate sperm passage. On the other hand, our investigations of in vitro sperm penetration ability have shown that spermatozoa with excellent motility could penetrate even relatively poor cervical mucus (unpublished data). Thus, in all 11 AD cases with conception in spite of reduced mucus crystallization, the sperm samples showed extremely high quality with good motility and an excellent penetration ability in the capillary migration test. n the investigation of semen property borderlines, only samples used for AD in women with a cervical mucus characterized by a degree of crystallization of were included. All these inseminations must have been performed near the time of ovulation, and the condition in the cervix was obviously good. Therefore, it could be concluded that each insemination had a reasonable opportunity to fertilize an ovum. When sperm count only is taken into consideration, the results make 20 million/ml the lower limit of "normal" fertility. This is in agreement with the findings of MacLeod and Gold, 2 but disagrees with the figure ofl0 million/ml reported in other studies 5 7 to be the lower limit. Sperm density is commonly used for the evaluation of fertilizing capacity. The results of the present study show that there was no statistically significant difference between density figures above 20 million/m!. The lowest density leading to conception was 15 million/m!. n this sample, however, both motility and morphologic characteristics were extremely good, and the in vitro penetration test showed a high grade of progressive motility. The unusual high conception rate of 22.7 in the group of samples with sperm density from 30 million/ml to 39 million/ml in this material can be explained by the fact that 18 inseminations using sperm from a single donor resulted in 50 of these conceptions. This donor had a relatively low sperm density but excellent sperm motility and a high percentage of sperm with normal forms. The high conception rate of this donor underlines motility as one of the most useful determinants of the fertilizing capacity. No reduction in conception rate was found among samples containing at least 50 normal spermatozoa. This percentage is lower than that of 60 declared by MacLeod and Gold 4 to be the lowest normal limit. Great variation, however, exists between laboratories in morphologic rating of spermatozoa. n this study all examinations were performed by the same experienced investigator. n this series one conception occurred with a sperm sample containing 50 abnormal sperm, and no pregnancy occurred when the samples contained more than 50 abnormal sperm. Determination of the quantitative and qualitative motility are important parameters for assessing male fertility potential but are usually evaluated from subjective calculation methods and are therefore not precise. The conception rate was significantly reduced in the present study when the percentage of motile sperm was below 50, and no pregnancy occurred when the percentage was less than 30. The remarkably high conception rate in the group of samples with 35 motile sperm probably reflects that 3 of the 5 pregnancies in this group followed 11 inseminations using sperm from a single donor with very high sperm density (176 to 203 million/m). For qualitative motility determination, the data indicate that the probability for conception was reduced when the degree of motility was grade 2 or less. Ulstein 1 reported that sperm penetration had a higher dis- Table 7. Conception Rate According to Degree of Motility Degree of No. of No. of Conception motility inseminations conceptions rate ;;: Edvinsson et a. Semen and cervical mucus at conception 331

6 Table 8. Conception Rate According to in Vitro Sperm Penetration Ability Following 369 nstances of AD Penetration No. of No. of Conception level inseminations conceptions rate mmlhr ;; < Total criminating power for fertility and infertility than other semen property. Our modification of the Kremer in vitro penetration test using egg white instead of cervical mucus has in this study shown a good correlation between penetration ability and conception rate. The reduced conception rate when the penetration level was less than 10 mmlhour indicates this level to be regarded as the lower limit of fertilizing probability. t has been previously reported that some semen properties are correlated with each other,1, 4, 12, 14 and that good motility and good morphologic characteristics can be compensating factors for low sperm density.4 Our data support this opinion. Probably a certain number of morpho logically normal sperm with good progressive motility are required for a reasonable chance of conception. The sperm penetration test has been shown to give reliable information on the progressive motility of the sperm and has been shown to be a useful instrument for the evaluation of the fertility potential. With our reading of the penetration test, a man who in at least two semen samples has a sperm density of more than 20 million/ml, more than 50 normal forms, and a penetration level of 10 mmlhour or more must be considered fertile. REFERENCES 1. Ulstein M: Sperm penetration of cervical mucus as a criterion of male fertility. Acta Obstet Gynecol Scand 51:335, MacLeod J, Gold RZ: The male factor in fertility and infertility.. Spermatozoon in 1000 men of known fertility and in 1000 cases of infertile marriage. J Urol 66:436, MacLeod J, Gold RZ: The male factor in fertility and infertility.. An analysis of motile activity in the spermatozoa of 1000 fertile men and 1000 men in infertile marriage. Fertil Steril 2:187, MacLeod J, Gold RZ: The male factor in fertility and infertility. V. Sperm morphology in fertile and infertile marriage. Fertil Steril 2:394, Santomauro AG, Sciarra JJ, Varma AO: A clinical investigation of the role of the semen analysis and postcoital test in the evaluation of male infertility. Fertil Steril 23:245, Rehan NE, Sobrero AJ, Fertig JW: The semen of fertile men: statistical analysis of 1300 men. Fertil Steril26:492, Zukerman Z, Rodriguez-Rigau LJ, Smith KD, Steinberger E: Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril 28:1310, Eliasson R: Standards for investigation of human semen. Andrologia (continues Andrologie) 3:49, Kremer J: A simple penetration test. nt J Fertil 10:209, Nilsson S, Edvinsson A, Steen Y: The egg white as medium in the sperm penetration test. Unpublished data 11. Quinlivan WLG, Sullivan H: The immunologic effects of husband's semen on donor spermatozoa during mixed insemination. Fertil Steril 28:448, Freud M: nterrelationships among characteristics of human semen and factors affecting semen-specimen quality. J Reprod Fertil 4:143, Read MD, Schnieden H: Variations in sperm count in oligozoospermic or asthenozoospermic patients. Andrologia 10:52, Makler A, tskovitz J, Brandes JM, Paldi E: Sperm velocity and percentage of motility in 100 normospermic specimens analyzed by the multiple exposure photography (MEP) method. Fertil Steril 31:155, Edvinsson et al. Semen and cervical mucus at conception Fertility and Sterility