Screening of karyotype and semen quality in an artificial insemination program: acceptance and rejection criteria

Transcription

1 FERTIUTY AND STERILITY Copyright c 1983 The American Fertility Society Vol. 40, No.5, November 1983 Printed in U.SA. Screening of karyotype and semen quality in an artificial insemination program: acceptance and rejection criteria Colin D. Matthews, M.D. ** Judith H. Ford, Ph.D.t John C. Peek, Ph.D.* Michael McEvoy, M.B.B.S.* University of Adelaide, Adelaide, and The Queen Elizabeth Hospital, Woodville, South Australia, Australia Semen quality and karyotype were screened in all men offering to be donors for an artificial insemination (AID) program. The criteria for accepting or rejecting semen have now been set with respect to this sample of the population. There was no evidence of differences between the pregnancy rates of the accepted donors. One of 172 potential donors with a clear medical history had a chromosomal abnormality,4 had pericentric inversions of chromosome 9, and 14 had other heterochromatic variants. Of the recipients of AID, 5 of 196 women had chromosomal abnormalities, and 12 had heterochromatic variants. Fertil Steril40:648, 1983 The practitioner of artificial insemination by donor (AID) has many and varied responsibilities, including duties toward the recipient couple, the child to be conceived, the donor, and society. In addition, the efficiency of AID is of prime concern, and consequently the practitioner wishes to select donors who are fertile and have high fecundity. In order to do this, some centers select only men who have already fathered children, l but most programs must accept men, usually students, whose fertility is unproven. Semen analysis then becomes the main criterion for assessing suitability. While there is evidence for some correlation between semen parameters and the likelihood of being fertile or infertile,2, 3 deciding upon the Received December 20,1982; revised and accepted July 27, *Reprint requests: Colin D. Matthews, M.D., Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia 5011, Australia. tdepartment of Cytogenetics. *Department of Obstetrics and Gynaecology. 648 Matthews et ai. Karyotype and semen quality in AID critical values for rejecting or accepting donors is largely arbitrary, and different centers have adopted different values. 1, 4-7 Given the possibility that semen characteristics may differ between populations and the fact that the measurement of some semen variables, motility, and morphology in particular, is partly subjective,s there may be merit in each center's deciding on its own criteria rather than having a common standard. Here we describe our criteria and the reasons for making these selections. It is generally accepted that some form of genetic screening of donors and recipients should take place, but the depth to which this is performed varies, and the necessity for the chromosomal examination of donor and recipient remains a controversial issue. In this study we karyotyped all potential donors and recipients in order to establish the incidence of chromosome abnormalities in these populations. It was our original belief that men with chromosomal abnormalities should not be used as sperm donors and that women with abnormalities should be offered prenatal diagnosis.

2 Has anyone in your family had any of the following conditions? Please include those family members who may have died. Think of your family as any brothers, sisters, father, mother, maternal and paternal aunts, uncles, and grandparents. Congenital Conditions Cleft palate or cleft lip Congenital heart disease Clubfoot Spina bifida Adult Conditions Mental retardation Epilepsy Premature senility Parkinsonism Tay-Sachs disease Schizophrenia Manic-depressive illness Lack of muscle coordination Huntington's chorea Myasthenia gravis If "Yes" to any, please answer: Specific relation: Conditions: Age affected: Hydrocephalus (water on the brain) Mongolism (Down's syndrome) Undescended testicles Dwarfism Blindness Color blindness Glaucoma Progressive kidney disease Neurofibromatosis Psoriasis Early deafness before 50 years Arthritis before 50 years Sickle cell anemia Hemophilia Cancer before 40 years Cirrhosis of the liver Stroke before 50 years Raised blood pressure before 50 years Heart attack before 50 years Cataracts before 40 years Diabetes before 30 years Cystic fibrosis Figure 1 Questionnaire for genetic history. MATERIALS AND METHODS SELECTION OF SUBJECTS Donors Two hundred sixteen men, most of them students, approached The Queen Elizabeth Hospital Fertility Clinic between 1974 and early 1982 to' he donors for AID. Twenty-three percent had fathered children. All donors were interviewed, and their medical and genetic history was assessed. Genetic historytaking was not structured, but rather a general inquiry was made using a questionnaire, in the hope of detecting any genetically related disease (Fig. 1). Particular emphasis was given to the detection of Huntington's chorea, dwarfism, Marfan's syndrome, cystic fibrosis, phenylketonuria, thalassemia, Tay-Sachs disease, renal disease, and congenital abnormalities. Screening normally included serum tests for venereal disease and hepatitis B antigen and, where appropriate, thalassemia and Tay-Sachs disease. Semen samples were obtained by masturbation at the clinic, after 3 to 7 days' abstinence, and were subjected to routine semen analysis. 9 Percentiles were interpolated from cumulative frequency curves. Cube root transformations of volume and sperm concentration were taken for statistical analysis; but proportions of motile sperm and sperm with normal morphology were left untransformed. Semen was diluted with egg-yolk citrate medium or a chemically defined medium based on Ham's F-10, and the mixture was vapor-frozen at C.lO Recipients Married couples were interviewed; and if AID was deemed suitable by the clinician and the couple, the female partner was routinely screened for blood type, serum prolactin, the presence of rubella antibodies, and karyotype. Additional investigations were performed if indicated. The general techniques of AID have already been described. l1 Vol. 40, No.5, November 1983 Matthews et al. Karyotype and semen quality in AID 649

3 FECUNDITY OF DONORS The pregnancy rate of a donor depends not only on the fecundity of the donor but also on the fecundity of the women for whom the semen is used. However, if each donor is used to inseminate several women, the differences in fecundity of the women will tend to average out, so that it is possible to compare the relative fecundity of donors. Since life-table analysis of AID pregnancy rates of recipients has indicated that only some women seem to be fertile under the conditions of AID,12 the present analysis was restricted to include only women who had had a previous pregnancy by AID or who ultimately became pregnant on the AID program. In addition, only cycles in which ovulation was proven (by luteal phase serum progesterone levels > 20 nmolll) and in which the fertile period was covered (as recognized by rapid luteinizing hormone monitoring ll ) were considered. The pregnancy rate of a donor equaled the number of cycles in which a pregnancy occurred, divided by the total number of cycles for which the donor was used. The proportions so obtained were tested for homogeneity.13 KARYOTYPING Chromosomes for karyotyping were prepared, from phytohemagglutinin-stimulated blood cultures and analyzed after Giemsa banding. Where significant heterochromatic "variants" were suspected, centromeric (C-) bands were also examined. Heterochromatic regions on auto somes were described as qh + if the size of the C-banded region was 1.5 times the size of a 16p in that cell. The Y chromosome was described as Y q + if it was at least as large as a chromosome 17, and the increase in size was due entirely to the C-banded region of the long arm. A variant of chromosome 9 was described as a "complete" pericentric inversion of 9qh if at least two thirds of the heterochromatin was located in the short arm, i.e., above the centromere. Partial pericentric inversions were either not recorded or recorded as "incomplete." In all, 172 donors and 196 recipients were karyotyped. REJECTION OF DONORS RESULTS The 216 potential donors had a median age of 24 years; the 5th and 95th percentiles were 18 and 42 years. Of the whole group screened, one man had a positive test for syphilis, one for gonorrhea, and another for hepatitis B antigen. These three men, who were excluded from the program, represented 1.4% of the total sample. Ten men (4.6%) failed to return to the clinic following their initial visit. Nine men (4.2%) were rejected because there was an adverse factor in their medical or family background. These factors included histories of neoplastic disease (five men) or schizophrenia (two men) in their immediate relatives; in addition, the presence of a cleft palate and juvenile diabetes was recorded in two subjects. Of the 136 men accepted as donors, 32 (23.5%) had some suspect factor(s) in their family genetic history and were accepted with some reservation. The most common reasons for rejection as a semen donor were either semen of poor quality or the failure of sperm to exhibit adequate motility after cryopreservation. The characteristics of the first semen sample from the 216 men are presented in Figure 2. Median values (and 5th and 95th percentiles) were 3.2 ml (1.1 to 6.8 ml) for volume, 68 x 106/ml (22 to 144 x 106/ml) for sperm concentration, and 58% (37% to 76%) for proportion of motile sperm. The year-by-year changes in semen variables were analyzed by analysis of variance. While there was no evidence of variation in volume or sperm concentration, the average motility fell progressively from 70% (n = 7) in 1974 to 54% (n = 47) in 1981, which was statistically significant (F[7,199] = 4.18, P < 0.05). Similarly, the proportion of sperm judged morphologically normal declined from 91% in 1974 to 55% in 1981, and this difference was highly significant (F[7,191] = 21.7, P «0.001). The median morphology in 1980 to 1981 was 57%; the 5th and 95th percentiles were 26% and 77%. The measurement of the proportion of motile sperm and the proportion with normal morphology is more subjective than that of volume and concentration. The average pregnancy rate of 28 donors who were used for eight or more cycles in women of proven fertility under the conditions of AID was = The individual pregnancy rates of these 28 donors were tested for homogeneity. The differences were not significant (X 2 = 29.61, df = 27), indicating that the pregnancy rates observed could have arisen solely from sampling from a population of men of uniform fecundity. 650 Matthews et al. Karyotype and semen quality in AID

4 Frequency o 8,. 8 L---I---'--..L.:::=i't-= o > qo o 20 qo Cumulative frequency ( ~ ) qo 20 L..C---'-----'-----'--Lt'f--J 8 >8 o , qo o 20 qo Semen volume (ml) Sperm concentration ( 106 I ml ) Motility ( % ) Normal morphology ( ~ ) Figure 2 Frequency distributions of semen characteristics of potential donors for AID (n = 216), except for normal morphology, for which only 1980 to 1981 (n = 104) data are shown. KARYOTYPING OF DONORS AND RECIPIENTS The results ofkaryotyping 172 potential donors are summarized in Table 1. Fourteen men (8.1%) demonstrated heterochromatic variants, 4 (2.3%) had pericentric inversions of chromosome 9, and 1 man had a possible abnormality of chromosome 1 with a duplication of band q21.1 (Fig. 3a). The remaining 153 men (89%) had a normal 46,XY constitution. The results of karyotyping 196 female recipients are summarized in Table 2. Twelve subjects (6.1%) demonstrated heterochromatic variants, the majority affecting chromosome 9, and 5 subjects (2.6%) had abnormal chromosomal constitutions, including one Robertsonian translocation t(14;22)(14qter~cen~22qter), two reciprocal trans locations (Fig. 3b and c), and two chromosomal deletions, one of which is illustrated (Fig. 3d). Vol. 40, No.5, November 1983 DISCUSSION This article principally deals with two of the many aspects that pertain to the selection of donors and recipients for AID,5,7 namely, semen quality and hidden genetic abnormalities. Since seminal characteristics of different populations may vary,14 and because the measurement of some semen variables may be dependent on the individual laboratory,8 we now base our criteria for accepting or rejecting donors on the semen quality of a cross-section of local men rather than on arbitrary values considered to be associated with high fecundity. The criteria for accepting a semen sample have now been set equal to the 30th percentiles of our reference population: sperm concentration ~ 50 x log/ml, ~ 50% motile sperm, and ~ 50% morphologically normal sperm. In addition, a semen sample must exhibit cryosurvival of ~ 50%, and potential do- Matthews et ai. Karyotype and semen quality in AID 651

5 Table 1. The Chromosomal Analysis of Potential Semen Donors 'Heterochromatic variants Normal karyotype Pericentrlc inversion 9 Other [inv(9)(pll;q13)] 46,XY Complete (n = 153) (n = 3)a Incomplete (n = 1) 1qh+ (n = 3) 1qh+,9qh+ (n = 2) 1qh+,15p+ (n = 1) 6qh+ (n = 1)a 9qh+ (n = 4) 16qh+ (n = 1) Yq + (n = 1) Yq+ + (n = 1)a Abnormal?dup(1) (q21.1)a afive potential donors were rejected because of their chromosomal findings (see text). nors are rejected if more than one of the first three samples has a cryosurvivalless than this value. The value of establishing local criteria can be readily appreciated. Half of all 216 men and 32% of the 100 men who approached the clinic in 1980 and 1981 (when average morphology was lower) were acceptable under the new criteria. If, however, the criteria of ~ 75 x 106 sperm/ml, ~ 50% motility, and ~ 50% cryosurvival6 had been applied, then only 13% of our potential donors would have been acceptable. By comparison, however, Leto and Frensilli6 found 55% of their potential donors acceptable under the above criteria. It remains uncertain whether true population differences underlie this finding. When an analysis of pregnancy rates of donors who were accepted for AID was performed, there, was no evidence that different donors had different pregnancy rates. Consequently, there was no justification for looking further for factors or qualities that might correlate with pregnancy rates. While all donors were interviewed and had possible genetic aspects of their family history assessed, only 4.2% of potential donors were rejected on this basis. Genetic screening by history is dependent on the level of competence and the depth of questioning. Timmons et ai.,15 using an extensive data interview and collection system, found that 5.4% of 149 donors had self-assessed genetic disorders, whereas extensive investigation revealed that 29.5% had conditions within the family which warranted further investigation, a 5.5-fold discrepancy. On the other hand, the study of Curie Cohen and associates16 showed that while 96% of 652 Matthews et al. Karyotype and semen quality in AID physicians do take a genetic history of their donors, much of it is subjective. Our own program is based on a thorough checklist of specific conditions, plus a medical (in contrast to genetic) interview, and thus may be regarded as closer to being "self-assessed." On this basis, the rejection rate was relatively low (4.3%) and similar to that of Timmons et ai. 15 While it is important to reject potential donors who may carry familial genetic defects, the value of karyotyping has been controversiai.5, 7, 17 Of the 172 men subjected to karyotyping in this study, 2 men whose fecundity was unproven had a complete peri centric inversion of chromosome 9, and 1 man whose fecundity was unproven had an exceptionally large Y chromosome (D size). These chromosomal findings are regarded as "variants" rather than abnormalities; nevertheless, these men were not accepted as donors because at the time of investigation these variants had been shown to be associated with histories of recurrent abortion.1s.21 A third man with a complete pericentric inversion of chromosome 9 was accepted as a donor because he and his wife had a good reproductive history. The incidence of complete peri centric inversions of chromosome 9 in the general population is ~ 0.6%22 which is lower than the 1.7% detected in this group, but not significantly so (P = 0.065). There is no obvious reason for the biased detection in this study, but the lack of detection in the female population suggests that there could be some sex-associated clustering. If the men and women are considered together with respect to this variant, our detection rate is 0.8%, which is close to the reported population frequency. A 6qh + variant was observed in one donor, and this region had not only the appearance and qual- Table 2. Chromosomal Analysis of Potential Recipients of AID Heterochromatic variants Normal karyotype lnv(9)(pll;q13) Other Abnormal 46,XX 0 9qh+ (n = 6) 46,XX,t(6;10) (n = 179) (qll.6;p15) 1qh + (n = 3) 45,XX,t(14;22) 22p + (n = 2) 15p + (n = 1) (14qte~ cen->22qter) 46,XX,t(14;19) (pl1;p13) 46,XX,del(13) (pll.1->pter) 46,XX!46,XX, del(20)(q2.11 ->qter)

6 c d,, ~ 1. I' I: f'., t! I cp, 46,XX. ti4;19hp14.p13 3! Figure 3 Partial karyotypes are shown for (a) chromosome 1, C-banded and G-banded, demonstrating a Giemsa and C-band negative band between two blocks of heterochromatin in the region q12; detected in a potential donor, G. T.; (b) reciprocal translocation between chromosomes 6 and 10 in recipient B. H.; (c) reciprocal translocation between chromosomes 4 and 19 in recipient C. P.; (d) deletion of chromosome 20 found in 25% of blood and skin cells of recipient M. P. ity of a 9qh + but had a high frequency of breakage. Since the usual C-band polymorphism on chromosome 6 is located in band 6pll,23 we suspected that this finding might represent a translocation; but we were unable to exclude this possibility with the preparations obtained. A decision was therefore made not to accept the subject as a donor at that time. A fifth potential donor was not accepted because of a?abnormal chromosome 1. In this man the region between the centromere and band q21 was large and appeared to be composed of three segments, two Giemsa-positive and one Giemsanegative (Fig. 3a). The two Giemsa-positive regions, which were also C-band-positive, probably represent a duplication of band q12 (equivalent to the variant 1qh + ), but the origin of the interfacvol. 40, No.5, November 1983 ing Giemsa-negative band is unknown. This has been provisionally interpreted as a duplication of band q21.1. This duplication does not seem to be associated with any phenotypic abnormality, and the significance of the finding is not understood. Karyotype investigation, therefore, was responsible for the rejection of 2.9% (5 of 172 men) of the men screened. Of the 196 recipients of AID who were screened by chromosomal analysis, 179 (91.9%) were found to be normal, 12 (6.1 %) had heterochromatic variants, and 5 (2.5%) were found to have structural chromosomal abnormalities (Table 2). One subject decided against AID on the basis of a reciprocal translocation between chromosomes 4 and 19, and a second subject with a Robertsonian translocation of chromosomes 14 and 22 has since had two early abortions following AID. The 2.6% incidence of chromosomal abnormalities in the recipient group seems high. Surveys of newborn populations by conventional techniques 24 showed an incidence of balanced chromosome translocations of 0.3%. In the recipient population we found three translocations (1.7%; significantly higher than 0.3%; P = 0.019), and all three of these could have been detected conventionally. Mosaicism is often undetected in surveys of the newborn because of the small number of cells analyzed. The 25% mosaicism of the deletion of the q arm of chromosome 20 may have escaped detection. The fifth abnormality, which involved the loss of the p arm and satellites of chromosome 13, is not classifiable as a variant. 25 However, this chromosomal region is often deleted in Robertsonian translocations and is unlikely to be of phenotypic significance. Although currently the occurrence of congenital abnormalities in children derived from AID programs is generally not greater than expected, considerable responsibility lies with the practitioner of AID to avoid the birth of a child with a congenital abnormality or of a child at risk for an inherited medical disorder prior to reaching the usual life span. Although the assurance given by an uneventful genetic history and normal chromosomal analysis is limited by the fact that the majority of genetic and congenital nongenetic abnormalities arise de novo, it is a clear responsibility of practitioners of AID to recognize those transmissible conditions with a predictable inheritance pattern. The role of karyotyping either donor or recipient will remain a controversial area, but judgmatthews et al. Karyotype and semen quality in AID 653

7 ment as to whether the investigation is worthwhile requires such data as indicated in this study. REFERENCES 1. Servoz GM: Organization of the French sperm banks C.E.C.O.S. In Instrumental Insemination: Clinical Andrology, Edited by ESE Hafez, K Semm. The Hague, Martinus Nijhoff, 1982, p David G, Jouannet P, Martin-Boyce A, Spira A, Schwartz D: Sperm counts in fertile and infertile men. Fertil Steril 31:453, Eliasson R: Analysis of semen. In The Testis, Edited by H Burger, D de Kretser. New York, Raven Press, 1981, p Hansen KB, Nielsen NC, Rebbe H: Artificial insemination in Denmark by frozen donor semen supplied from a central bank. Br J Obstet Gynaecol 86:384, Johnston I: The donor. In Artificial Insemination by Donor, Edited by C Wood. Melbourne, Brown Prior Anderson Pty. Ltd., 1979, P Leto S, Frensilli FJ: Changing parameters of donor semen. Fertil Steril 36:766, Hermanns U, Hafez ESE: Selection and assessment of donor. In Instrumental Insemination: Clinical Andrology, Edited by ESE Hafez, K Semm. The Hague, Martinus Nijhoff, 1982, p Freund M: Standards for the rating of human sperm morphology. Int J Fertil 11:97, Eliasson R: Standards for investigation of human semen. Andrologia (continues Andrologie) 3:49, Peek JC, Gilchrist SJ, Kelso CM, Quinn PJ: Comparison of three cryoprotective solutions for human semen. Clin Reprod Fertil 1:301, Matthews CD: Artificial insemination-donor and husband. In The Infertile Couple, Edited by RJ Pepperell, B Hudson, C Wood. Edinburgh, Churchill-Livingstone, 1980, p Peek JC, Godrey B, Matthews CD: Evaluating the effectiveness of AID by extention of lifetable analysis. In Proceedings of the Annual Meeting of the Fertility Society of Australia, Vol 1, September 30 to October 2, Abstract Snedecor GW, Cochran WG: Statistical Methods. Ames, Iowa, Iowa State University Press, Pryor JP: Seminal analysis. Clin Obstet Gynaecol 8:571, Timmons MC, Rao KW, Sloan CS, Kirkman HN, Talbert LM: Genetic screening of donors for artificial insemination. Fertil Steril 35:451, Curie-Cohen M, Luttrell L, Shapiro S: Current practice of artificial insemination by donor in the United States. N Engl J Med 300:585, Danks DM: Genetic considerations. In Artificial Insemination by Donor, Edited by C Wood. Melbourne, Brown Prior Anderson Pty. Ltd., 1979, P Boue J, Taillemit JL, Hazael-Massieux P, Leonard C, Boue A: Association of pericentric inversion of chromosome 9 and reproductive failure in ten unrelated families. Hum Genet (continues Humangenetik) 30:217, Patil SR, Lubs HA: A possible association of long Y chromosomes and fetal loss. Hum Genet 35:233, Ford J: Cytogenetics of infertility and habitual abortion. Rec Adelaide Child Hosp 1:287, Genest P: Chromosome variants and abnormalities detected in 51 married couples with repeated spontaneous abortions. Clin Genet 16:387, Muller HK, Klinger HP, Glasser M: Chromosome polymorphism in a human newborn population. Cytogenet Cell Genet 15:239, Madan K, Bruinsma AH: C-banded polymorphism in human chromosome No 6. Clin Genet 15:193, Jacobs PA, Frankiewicz A, Law P: Incidence in mutation rates of structural rearrangement of the autosomes in man. Ann Hum Genet 35:301, Nielsen J, Friedrich U, Hreidousson BA: Frequency of deletion of short arm satellites and acrocentric chromosomes. J Med Genet 11:177, Matthews et al. Karyotype and semen quality in AID