Sex selection by sperm separation and insemination*

Transcription

1 FERTILITY AND STERILITY Copyright 1984 The American Fertility Society Printed in U.8A. Sex selection by sperm separation and insemination* Stephen L. Corson, M.D. thil Frances R. Batzer, M.D.:j: 11 Nancy J. Alexander, Ph.D.~ Sheldon Schlaff, M.D. ** Chari Otis, M.S. II Philadelphia Fertility Institute, Pennsylvania Endocrine Laboratory, Pennsylvania Hospital, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, and Oregon Health Sciences University, Portland, Oregon The Ericsson albumin filtration technique was used to collect a fraction rich in Y sperm for selective insemination in couples desiring a male infant. Of 35 conceptions in which sex was known at delivery or spontaneous abortion, there were 28 males (80%). Twelve pregnancies were achieved after separation of sperm in a Sephadex gel filtration system designed to allow for collection of a fraction enriched in X sperm. Seven pregnancies have resulted in females, two in males, and one in twins of each sex. One patient aborted, and one is still pregnant. While selection for either sex can be done electively, on the basis of sociologic preference, female selection has, as an additional indication, avoidance of male offspring to carriers of sex-linked diseases. Fertil Steril 42:756, 1984 Prospective investigation of the effect of various treatments on the X/Y sperm ratio in the ejaculate was possible after discovery of a reliable method for "Y" body staining. 1, 2 Ericsson et al, 3 in 1973, reported a method of isolation of a seminal fraction rich in Y sperm after treatment with bovine serum albumin columns. Separation was said to be a consequence of Y sperm having Received April 9, 1984; revised and accepted July 24, *Presented in part at the Fortieth Annual Meeting of The American Fertility Society, April 2 to 7, 1984, New Orleans, Louisiana. treprint requests: Stephen L. Corson, M.D., Philadelphia Fertility Institute, 330 South 9th Street, Philadelphia, Pennsylvania :j:philadelphia Fertility Institute. Pennsylvania Hospital. IIDepartment of Obstetrics and Gynecology, University of Pennsylvania School of Medicine. ~Oregon Health Sciences University. **Pennsylvania Endocrine Laboratory. "greater ability to penetrate an interface between liquids and between continuous gradients of an isolation medium." Recovery rates were found to be between 30% and 40% of the initial total sperm number. Some investigators 4,5 were unable to duplicate Ericsson's results with respect to separation or recovery rates. Dmowski et al 6 found a significant increase in sperm motility, progressive drive, and Y-bearing sperm, as well as a percentage increase in normal forms after albumin treatment. In addition, fathers of three or more daughters had significantly fewer Y -bearing sperm (prior to separation) than did control subjects. Ten conceptions occurred; five sons and two daughters were delivered, and there was one spontaneous loss of a male fetus and two ongoing pregnancies at the time of the report. Following a description by Steeno et al,7 Quinlivan et al 8 used a Sephadex (Pharmacia, Piscataway, NJ) gel filtration method to collect frac- 756 Corson et al. Sex selection

2 tions rich in X-bearing sperm. We have successfully used this technique in clinical application. 9 In this article we describe the use in a clinical setting of both the albumin and the Sephadex filtration techniques to select for male or female offspring. MATERIALS AND METHODS Patients reported in this article were treated by identical seminal protocol and clinical timing of insemination in the two centers involved in the study. MALE SELECTION The Ericsson method 3 was used to select for male offspring in 79 women. Human serum albumin was used rather than bovine serum albumin. Forty women conceived, but complete data sufficient for statistical analyses of seminal factors were available for only 30 women. The original Ericsson three-step procedure was used for all couples except two, in whom low sperm densities necessitated a two-step process. Insemination timing was determined by the appearance of copious, clear, and elastic cervical mucus during daily serial examination at the time ovulation was expected on the basis of previous basal body temperature (BBT) charts. In addition, for some patients rapid luteinizing hormone (LH) determinations were made, and insemination was performed on the day of or on the day after the LH was ~ 50 miu/ml. Conception occurred in two patients who received clomiphene citrate (CC) because of anovulation. Both patients received 100 mg daily on cycle days 5 through 9. Human chorionic gonadotropin (hcg), 5000 U, at the time of insemination, was given to 11 of those who conceived. FEMALE SELECTION Sperm were separated with a Sephadex gel filtration technique in order to select for female offspring in 19 women. Inseminations were timed as with patients treated for male selection. CC, 100 mg daily for 5 days, was administered to one patient and periovulatory hcg, 5000 U, was given to two other patients. In all instances, for both male and female selections, couples were asked to abstain from coitus for the 4 or 5 days preceding planned insemination; otherwise, each couple used mechanical methods of contraception until the BBT had been elevated for 3 consecutive days. The choice of insemination technique, intracervical or intrauterine, depended on the number of recovered sperm and the number of leukocytes in the ejaculate. Statistical methods of analysis included linear regression techniques, chi-square analysis, Student's t-test between means, and binomial distribution probability analysis. MALE SELECTION RESULTS There were 40 conceptions in the 79 patients inseminated with this technique designed to enhance the chance for conception of a son. There were 28 live male births, 3 spontaneous abortions (sex unknown), 6 female births, 1 female secondtrimester spontaneous loss, and 1 molar pregnancy. One patient is still pregnant. More sons were born than would be expected by chance (P < 0.005). Taking into account the 26 patients on whom we had complete clinical and laboratory data (19 males17 females), we found a total of 55 previously delivered daughters and only 2 sons. Data on 19 pregnancies resulting in delivered sons, 6 pregnancies resulting in delivered daughters, 1 pregnancy resulting in an aborted female fetus, and 43 nonconceiving couples (groups 1 to 3) were analyzed statistically. Three early abortions and a molar pregnancy were excluded from further analysis. Nine other couples (who had sons) were excluded from analysis because of incomplete clinical or seminal data. The mean time for conception with treatment was 2.2 cycles for those having sons and 1.7 cycles for those having daughters; the mean number of cycles for those not conceiving was 2.8. Maternal ages were 31.5, 31.3, and 34.1, respectively. Nonconceivers were statistically older than conceivers (P < 0.005). There was no difference among the three groups in the interval between the last delivery and entry into the program. Values for semen volume, sperm concentration, and total number of motile sperm are presented in Table 1. The mean preselection semen volume for specimens that resulted in a son was 3.0 ml; the difference between this value and values for specimens from men siring daughters and from men not causing pregnancies was of borderline statistical significance (P < 0.10). No differences in seminal Corson et al. Sex selection 757

3 Table 1. Male Sex Selection Males Conceiving Females Preseparation seminal values No Semen volume (mil 2.96 (0.40)a 4.06 (0.28) Sperm concentra 128 (27) 138 (18) tion x 10 6 Total motile 249 (46) 418 (66) sperm x 10 6 Postseparation seminal values Total motile 21 (l.9) 46 (12) sperm x 10 6 % Recovery of 12.3 (2.0) 11.0 (2.5) motile sperm Nonconceiving (0.28) 136 (12) 403 (58) 26 (3) 9 (0.8) athe values in parentheses are the standard error of the mean. concentration were noted among the groups, but men producing female offspring had higher total motile sperm counts prior to separation than men producing male offspring (P < 0.01). Mean motilities were 74%, 74%, and 71%, respectively, for groups 1 to 3 prior to separation and 82%, 84%, and 86%, respectively, after albumin column treatment. Table 1 shows the total motile sperm recovery after separation and the percentage of total motile sperm recovered. Although there were no statistical differences among the recovery rates for groups 1 to 3, fewer total motile sperm were recovered from men siring males than for those producing females (P < 0.01). Of 19 males, 8 were conceived with intrauterine insemination; of 7 females, 3 were conceived with this approach. Both women receiving CC delivered sons. Of 26 women conceiving, 11 received peri ovulatory hcg as an ovulation-stimulating adjunct. Six had sons, and five had daughters. The ratio was 13:2 in those not having hcg. Analysis involving the binomial theorem showed P < 0.05 for the difference of the distribution. No congenital abnormalities were observed in any of the offspring. FEMALE SELECTION Of the 19 women inseminated with sperm separated by the Sephadex filtration method, 12 conceived. Sixteen of the couples entered into treatment because of preference; 1 couple wished to enhance their chance of having a daughter because the woman was a probable carrier of Duchenne's muscular dystrophy. This woman had a very early spontaneous abortion. Four patients delivered a normal daughter, one had a daughter with a cardiac septal defect, and one set of male/ female twins was delivered in conjunction with CC treatment to stimulate ovulation. Amniocentesis in two patients who have not yet delivered demonstrated a normal XX karyotype; a third is still pregnant. Two patients delivered normal males for an overall result of seven daughters, two sons, and one set of mixed twins to date. Of the 12 patients conceiving, 3 were nulligravida; the other 9 had a total of 15 previous children, all of whom were sons. In those not pregnant, two had husbands with sperm showing low motility and leukocytes in the ejaculate. Three of the nonconceiving women received CC for anovulation. Pregnancy was achieved in 1. 7 cycles of care versus 2.5 cycles for those who left the program. Maternal age was 33.7 versus 32.5 for dropouts. Table 2 shows seminal data. The mean volume of semen from men who caused conceptions was greater (4.6 ml versus 2.8 md than the volume from those who did not; but the difference was not of statistical significance. Similarly, there was no statistical difference in the total number of motile sperm or the semen concentration between those who caused conceptions and those who did not. Table 2 also shows the postseparation seminal data. The number of motile sperm recovered and used for insemination was larger in the group that caused conceptions than in the group that did not, but the difference was not of statistical significance. The procedure for enhancing recovery of X sperm gave greater sperm recovery than the procedure for enhancing Y sperm recovery. DISCUSSION Hormonal levels may change the sex ratio; there has been a reported decrease in the num- Table 2. Female Sex Selection Conceiving Preseparation seminal values No. 12 Semen volume (md 4.06 (O.56)a Sperm concentra 72 (6.7) tion x 10 6 Total motile sperm 205 (39) x 10 6 Postseparation seminal values Total motile sperm 86 (15.3) x 106 0/0 Recovery of mo 42.7 (5.8) tile sperm Nonconceiving (0.37) 58 (9.9) 125 (26) 54 (13.2) 51.6 (8.6) athe values in parentheses are the standard error of the mean. 758 Corson et a1. Sex selection

4 Table 3. Sex Ratio of Offspring of Mothers Who Ingested Clomid (2369 Pregnancies of Investigational Patients from r Category Mothers Offspring Sex of offspring Male Female Male:female Unknown or ratio (of known unreported sex) Single pregnancies total (in :1.00 eluding stillbirth and abortion) Single pregnancies, live :1.00 births (including neonatal deaths) Single plus multiple preg :1.00 nancies total (including stillbirth and abortion) Multiple pregnancies total b 62 b 246 b 1.58:1.00 (incl uding stillbirth and abortion) Twin pregnancies b 36 b 238 b 1.55:1.00 Triplet pregnancies C 11 3c C 1.73:1.00 Quadruplet pregnancies c 7c 4c 2.43:1.00 Quintuplet pregnancies c 8c 1C 0.75:1.00 adata from Merrell Dow Pharmaceuticals, Inc., Cincinnati, OH, April bdata from computer print-out, not confirmed by case report review. CData from case report review. I ber of males to 43.5% when pregnancy has been initiated with CC or human gonadotropins.lo Sampson et al. 1l noted that with artificial insemination involving donor semen (AID) 46.1% of 89 CC-induced pregnancies resulted in sons, and 60.6% of 162 non-cc-induced pregnancies resulted in sons (P = 0.05). Corson et al. 12 did not observe a decrease in the number of males in a study in which pregnancies were induced with CC alone and with CC + hcg at midcycle: the male/female ratios for those two regimens were 29:24 and 59:51, respectively. In a subgroup of CC-treated AID patients, there were 24 males and 23 females. The largest known data collection of CC-induced pregnancies comes from the investigational files of Merrell Dow Pharmaceuticals, Inc., Cincinnati, OH. Data in Table 3 were prepared for us by Dr. Holtkamp.13 Although the 2369 pregnancies were conceived under a variety of conditions with respect to coitus versus husband or donor insemination, there is no change in the expected sex ratio except for multiple pregnancies in which there is actually a shift toward more males. Therefore, the reports demonstrating a shift to female offspring in association with CC use in the female may represent aberrations caused by small sample size. Insemination prior to or at ovulation was said by Kleegman 14 to produce an excess of male births. In Guerrero's studies/ 5, 16 BBT-timed insemination at ovulation produced 60% males, but coitus at ovulation resulted in 46% males. Other investigators, such as Harlap,17 have suggested that coitus after ovulation is more likely to result in a male birth. These data are confusing and often contradictory. Moreover, the reference point of ovulation in most of these studies was the thermal nadir of the BBT chart, which has been shown to be unreliable. lb, 19 Therefore, a more active approach, such as separation of the sperm and insemination based on LH levels, seems warranted. MALE SEPARATION Our experience in using sperm separation to enhance collection of Y sperm has resulted in 28 live male births from 35 conceptions with live deliveries or abortion with fetai sex known (80% male). The rate compares favorably with the documented group statistics achieved by eight centers in the United States that use the Ericsson technique: 93 males and 28 females. 20 Even though the mean age of females in the study population was over 31 years, pregnancy was achieved within two cycles for most. The mean age of the group that did not conceive was statistically higher. Only two women were treated with CC, and they both had sons. On the other hand, ovulation stimulation with hcg in 11 women abolished any discernible shift in the sex ratio. Since hcg was used to induce ovulation in the absence of an LH elevation, the effect may have Corson et al. Sex selection 759

5 been due not to hcg itself, but to variation in sperm exposure time within the female reproductive tract prior to fertilization. Because the sperm recovery rates were low (- 10.0%) and the mean of total motile sperm was 26 x 10 6, the full three-step Ericsson technique must necessarily be restricted to men with reasonably good semen volume, concentration, and sperm motility. Pregnancy was achieved with insemination of as little as 6 x 10 6 sperm. The Ericsson albumin column sperm separation method for enhancing the Y population seems to be efficacious and safe; only one congenital anomaly (a heart valvular defect) was observed in the series of 121 deliveries from the eight participating centers. FEMALE SEPARATION Separation of sperm to obtain a fraction rich in X sperm is a new procedure. Quinacrine staining of sperm prior to and after separation with a Sephadex column demonstrates an enrichment of X-bearing sperm. 9 These studies were carried out as a preliminary step in efforts to bring this technique to clinical use. They indicate that separation for an X-enriched fraction is as efficient as that for a Y -enriched fraction, and this will be the subject of a later communication. Although one may select female offspring for elective reasons, or to balance a perceived disproportion within a family unit, it is important to note that sex selection affords a method of dealing with sex-linked disease. The Sephadex separation results in greater total sperm recovery than the albumin column technique; however, the albumin column is much more effective in removing leukocytes, debris, and nonmotile sperm. Even though seminal prostaglandins are removed with both methods, it seems prudent to use the Sephadex-separated specimen with caution when planning intrauterine insemination. REFERENCES 1. Zech L: Investigation of metaphase chromosomes with DNA binding fluorochromes. Exp Cell Res 58:463, Barlow D, Vosa CG: The Y chromosome in human spero matozoa. Nature 226:961, Ericsson RJ, Langevin CN, Nishino M: Isolation of fractions rich in human Y sperm. Nature 246:421, Evans JM, Douglas TA, Renton JP: An attempt to separate fractions rich in human Y sperm. Nature 253:352, Ross A, Robinson JA, Evans HJ: Failure to confirm separation of X and Y bearing sperm using BSA gradients. Nature 253:354, Dmowski WP, Gaynor L, Rao R, Lawrence M, Scommegna A: Use of albumin gradients for X and Y sperm separation and clinical experience with male sex preselection. Fertil Steril 31:52, Steeno 0, Adimoelja A, Steeno J: Separation of X and Y bearing human spermatozoa with the Sephadex gel infiltration method. Andrologia 7:95, Quinlivan WLG, Preciado K, Long TL, Sullivan H: Separation of human X and Y spermatozoa by albumin gradients and Sephadex chromatography. Fertil Steril 37:104, Corson SL, Batzer FR, Schlaff S: Preconceptual female gender selection. Fertil Steril 40:384, James WH: Gonadotropins and the human secondary sex ratio. Br Med J 281:711, Sampson JH, Alexander NJ, Fulgham DL, Burry KA: Gender after artificial induction of ovulation and artificial insemination. Fertil Steril 40:481, Corson SL, Batzer FR, Otis C, Corson J: Clomiphene citrate: nuances of clinical application. Clin Reprod Fertil 2:1, Holtkamp DE: Personal communication, Kleegman SJ: Therapeutic donor insemination. Fertil Steril 5:7, Guerrero R: Sex ratio: a statistical association with the type and time of insemination in the menstrual cycle. Int J Fertil 15:221, Guerrero R: Type and time of insemination, menstrual cycle and human sex ratio. N Engl J Med 291:1056, Harlap S: Gender of infants conceived on different days of the menstrual cycle. N Engl J Med 300:1445, Hoff JD, Quigley ME, Yen SSC: Hormonal dynamics at midcycle: a reevaluation. J Clin Endocrinol Metab 57:792, World Health Organization, Task Force Investigators: Temporal relationships between ovulation and defined changes in the concentration plasma estradiol-1713, luteinizing hormone, follicle-stimulating hormone and progesterone. Am J Obstet Gynecol 138:383, Ericsson R: Gametrics Bulletin, January Corson et al. Sex selection