Sharing of human leukocyte antigens in couples with unexplained infertility affects the success of in vitro fertilization and tubal embryo transfer

Sharing of human leukocyte antigens in couples with unexplained infertility affects the success of in vitro fertilization and tubal embryo transfer Hong-Nemg Ho, MD: Yu-Shih Yang, MD, PhD: Rhong-Phong Hsieh, BS,b Heng-Ru Lin, BS: Shee-Uan Chen, MD: Hsin-Fu Chen, MD: Su-Cheng Huang, MD: Tzu-Yao Lee, MD, PhD: and Thomas J. Gill III, MDc

Taipei, Taiwan, Republic of China, and Pittsburgh, Pennsylvania OBJECTIVE: The purpose was to test further our hypothesis that genes, or genetic defects, linked to the major histocompatibility complex affect reproduction by correlating human leukocyte antigen sharing with the success or failure of in vitro fertilization and tubal embryo transfer in couples having unexplained infertility. STUDY DESIGN: Seventy-six couples with unexplained infertility who failed superovulation and intrauterine insemination at least three times were typed for human leukocyte antigens and treated by in vitro fertilization and tubal embryo transfer. The results were correlated with the sharing of human leukocyte antigens in the couples. RESULTS: Thirty-four of the women had successful pregnancies, 36 did not become pregnant, and six became pregnant but aborted shortly thereafter. There was a highly significant excess of human leukocyte antigen sharing in the couples who failed treatment: three of the A, B, DR, and DO antigens (p = 0.015) or two of the B, DR, and DO antigens (p = 0.015). No specific human leukocyte antigen alleles were present in excess. CONCLUSIONS: Genes, or genetiC defects, linked to the major histocompatibility complex significantly affect the success of in vitro fertilization and tubal embryo transfer just as they affect the prevalence of recurrent spontaneous abortion, cancer, and congenital anomalies. It appears as if the critical genes, or genetiC defects, are located in the B-DR-DO region of the major histocompatibility complex. (AM J OBSTET GVNECOL 1994;170:63-71.)

Key words: Unexplained infertility, in vitro fertilization and tubal embryo transfer, major histocompatibility complex-linked genes, reproductive immunogenetics Approximately three quarters of fertilized ova do not progress to a viable fetus, approximately 0.5% of pregnancies are repeatedly lost without any discernible reason, I and an average of 20% of couples in clinic-based studies have unexplained infertility.2 The factors influencing fertilization, implantation, and embryonic development must be understood to develop effective procedures for the treatment of such patients. The most effective current treatment involves assisted reproduc-

From the Departments of Obstetrics and Gynecology" and Clinical Pathology/ School of Medicine and Hospital, National Taiwan University, and the Department of Pathology, University of Pittsburgh School of Medicine.' Supported by grants from the National Science Council of the Republic of China (NSC82-0115-B002-498), The Department of Health Executive Yuan (DOH81-TD-004 and DOH82-TD-096), and the Pathology Education and Research Foundation. Received for publication May 6, 1993; revised July 14, 1993; accepted July 30, 1993. Reprint requests: Thomas J. Gill1l1, MD, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378194 $1.00 + .20 6/1/50374

tive procedures," and about one half of the patients so treated have successful pregnancies. 4 • 6 Unsuccessful treatment has been ascribed to failure of embryo transfer, fertilization, or implantation. 7 - 9 We have been interested in the genetic basis of reproductive failure of unknown cause, and our studies in experimental animals 10. 11 and in humans l '- 15 have led us to develop the hypothesis that genes, or genetic defects, linked to the major histocompatibility complex influence growth, reproduction, and susceptibility to cancer. I, 16-18 Our clinical studies have been performed in an ethnically homogeneous Chinese population in Taiwan, and they showed that there is an excess of human leukocyte antigen (HLA) sharing in couples in whom the woman develops a gestational trophoblastic tumor l ' and in couples having recurrent spontaneous abortions. 13, 14 In addition, there was an increase in recurrent spontaneous abortions, congenital anomalies, and cancer in the first-, secondo, and third-degree relatives of these couples. 15 The current study was undertaken to determine whether major histocompatibility complex-linked 63

64

Ho et al.

genes, or genetic defects, could also affect the outcome of assisted reproduction with in vitro fertilization (IVF) and tubal embryo transfer. It was performed in the same Chinese population in Taiwan in which our previous studies were performed. The results of this investigation should add more evidence to our hypothesis for the importance for the reproductive process of genes in the meyor histocompatibility complex-linked region and may provide a useful parameter for assessing couples for assisted reproductive therapy. Material and methods

Patient selection. Seventy-six couples with unexplained infertility were enrolled in this study between September 1989 and March 1993; the study was conducted at the Infertility Special Clinic of the Department of Obstetrics and Gynecology at the National Taiwan University Hospital. All the couples had normal results in a thorough infertility study'-6: repeated semen analyses, documentation of ovulation, hysterosalpingography or laparoscopic examination, postcoital testing, complete endocrine survey, ultrasonographic folliculometry, and, in some cases, analysis for antisperm antibodies by Immunobead assay and the zona-free hamster egg sperm penetration assay. The women in each of these couples had superovulation and intrauterine insemination for at least three cycles before entering the IVF and tubal embryo transfer program. When entering this program, all of the couples were typed for HLA antigens as described previously. 12. 13 Fifty-one normally fertile couples who had given birth to at least two children and had no history of abortion were used as the control group. 12, 13 In vitro fertilization and tubal embryo transfer. All of the women ovulated after treatment by one of three stimulation protocols. 4 -6 The specific protocol was chosen on the basis of each woman's response to her previous treatment by superovulation and intrauterine insemination, and previous studies 6 , 19 have shown that each of the three protocols has similar fertilization, implantation, and pregnancy rates. The three protocols are (1) clomiphene citrate (Clomid, Shionogi, Japan) and human menopausal gonadotropin (hMG) (Pergonal, Serono, Italy), (2) follicle-stimulating hormone (FSH) (Metrodin, Serono) and hMG, and (3) gonadotropin-releasing hormone analog (Decapeptyl, Ferring, Germany), FSH, and hMG. The ovarian response was monitored by transvaginal ultrasonography (Aloka SSD-620, Japan) and by daily serum estradiol, progesterone, and luteinizing hormone levels. Ten thousand international units of human chorionic gonadotropin (Pregnyl, Organon, The Netherlands) was given when at least two follicles were ~ 18 mm in diameter and the serum estradiol levels were adequate. Ultrasonographically guided transvaginal oocyte re-

January 1994 Am J Obstet Gynecol

trieval was performed 34 to 36 hours after human chorionic gonadotropin injection. The oocytes were cultured in human tubal fluid medium 20 supplemented with 10% heat-inactivated patient's serum. Before insemination the oocytes were incubated at 37° C in a humidified atmosphere of 5% carbon dioxide in air for 3 to 8 hours. Semen was obtained by masturbation 2 to 3 hours before insemination, and the motile sperm were collected by means of a "swim-up" technique. 21 Motile sperm (0.1 X 106 ) were added to each oocyte, and 16 to 18 hours later fertilization was documented by visualization of two pronuclei after the cumulus surrounding the oocyte was removed by gentle pipetting. If fertilization had occurred, the zygotes (usually :5 4) were transferred to fresh medium; other zygotes were frozen for future use. When the embryos reached the two- to four-cell stage (approximately 48 hours after oocyte retrieval), tubal embryo transfer was performed by laparoscopy under general anesthesia by means of a transfer catheter (GIFT No. 1010, William A. Cook, Melbourne, Australia) inserted into the fallopian tube and containing the embryos in 20 to 30 ILl of culture medium. In the clomiphene citrate-hMG and FSH-hMG protocols the luteal phase was supported by the daily intramuscular administration of 25 mg of progesterone in oil beginning on the day of embryo transfer and continuing for 14 days. In the gonadotropin-releasing hormone ana10g-FSH-hMG protocol 1500 IU of human chorionic gonadotropin was given on days 2, 5, and 8 after embryo transfer, and 50 mg of progesterone was given for 14 days beginning on the day of embryo transfer. Clinical pregnancy was determined by ultrasonographic visualization of the gestational sac(s) and an elevated serum level of human chorionic gonadotropin. Statistical analyses. The patients were separated into two groups: 34 successful pregnancies and 36 pregnancy failures. Six women became pregnant but aborted within the first 3 months, so they were not counted in the analysis of the success or failure of IVF-tubal embryo transfer. (An analysis counting them as failures was also performed, and the results of the analysis did not change). The comparisons of age, duration of infertility, number of oocytes recovered, oocyte fertilization rate, implantation rate, and HLA sharing were performed with the one-tailed Fisher exact test in which p :5 0.05 was taken as the level of significant difference. Results

The ages, obstetric histories, duration of infertility, and number of oocytes retrieved, fertilized, and transferred of the 34 women who had a successful pregnancy after IVF-tubal embryo transfer (Table I) and the 36 women who did not (Table II) were quite similar. The

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65

Table I. The age, duration of infertility, and results of IVF and tubal embryo transfer in women who were delivered after treatment

Patient

Age (yr)

Obstetric history*

Duration of infertility (yr)

No. of oocytes retrieved

No. of oocytes fertilized

No. of embryos transferred

No. of embryos implanted

No. of babies born

Sl S2 S3 S4 S5 S6 S7 S8 S9 SlO Sl1 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34

39 38 33 27 27 33 34 36 35 28 31 22 41 34 39 27 38 32 29 37 36 25 34 35 37 36 30 36 32 33 36 32 37 33

GOPO GOPO G1A1 GOPO GOPO GOPO GOPO G1A1 G1A1 GOPO GOPO GOPO G2A2 GOPO GOPO GOPO GOPO G1A1 GOPO G1P1 GOPO GOPO GOPO GOPO GIP1 GOPO GOPO GOPO GOPO GIAI GOPO GIP1 GOPO GOPO

2.5 5 3 3 4 5 2 2 3 2 5 3 2.5 3 9 2.5 6 2.5 2 6 2 4 4 2 3 6 5 6 6 4 6 3 7 5

6 7 7 4 4 9 8 4 10 3 6 5 5 4 5 5 6 14 11 8 21 9 3 7 2 11 7 12 15 7 12 15 12 6

4 7 4 3 4 6 4 3 5 2 5 3 3 4 3 4 6 5 5 5 12 7 2 5 2 10 1 9 8 4 9 14 11 6

4 4 4 3 4 3 4 3 5 2 4 3 3 4 3 4 4 5 5 5 4 4 2 5 2 4 1 4 4 4 5 4 5 4

1 2 2 1 2 1 2 1 1 1 1 1 1 2 1 1 2 1 2 2 2 2 1 1 2 1 1 1 3 1 1 1 1 2

1 2 1 1 2 1 2 1 1 1 1 1 1 2 1 1 1 1 2 2 2 2 1 1 1 1 1 1 2 1 1 1 1 1

*G, Gravida; P, para; A, spontaneous abortion.

HLA typing data for these two groups of women are shown in Tables III and IV. The HLA typing data for six women who had successful IVF and tubal embryo transfer but who aborted in the first trimester are given in Table V. Their HLA sharing pattern is the same as that ofthe group that failed to implant (Table VI) (e.g., 50% of these couples shared three or more HLA-A, B, DR, DQ antigens and two or more HLA-B, DR, DQ and HLA-A, B, DR antigens). There was a highly significant excess of HLA sharing in the couples who failed treatment by IVF and tubal embryo transfer compared with couples in whom the treatment was successful or with normally fertile couples (Table VI). There was no significantly increased sharing of any specific HLA allele at a given locus but rather an increased sharing of three of the HLA-A, B, DR, DQ antigens (p < 0.015 compared with tubal embryo transfer failures and p = 0.021 compared with normal controls) or two of the HLA-B, DR, DQ antigens (p = 0.015 in both cases). There was no significant increase in the

sharing of the HLA-A, B, DR antigens, however. The antigen distribution among the various HLA loci was the same in the successfully treated couples and in normally fertile couples. In Table VI the significance of the differences among those with success with tubal embryo transfer, those with failure with tubal embryo transfer, and normally fertile groups is given for each HLA locus. If the question is asked whether there is any significant sharing of antigens at a specific HLA locus, the probability of sharing at that locus plus the probabilities of sharing at the other four loci must be considered together (i.e., the probability of all five events happening together must be considered). This calculation is accomplished by means one of Bonferroni's inequalities. 22 If the probability of the event's happening is chosen as 95%, the p value for one of five events (i.e., HLA sharing at a particular locus) would have to be S 0.01 for it to be significant. Another way to approach this correction is to multiply the potentially significant p value by 5 to see

66

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January 1994 Am J Obstet Gynecol

Table II. The age, duration of infertility, and results of NF and tubal embryo transfer in women who failed to conceive Patient

Age (yr)

Obstetric history*

Duration of infertility (yr)

No. of oocytes retrieved

No. of oocytes fertilized

No. of oocytes transferred

F1 F2 F3 F4 F5 F6 F7 F8 F9 FlO F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24 F25 F26 F27 F28 F29 F30 F31 F32 F33 F34 F35 F36

33 34 32 38 38 28 34 28 40 34 35 36 30 33 28 32 26 32 27 34 42 31 32 39 34 32 34 35 35 39 31 30 41 32 32 32

GOPO GOPO GOPO GOPO GOPO G1A1 GOPO GOPO GOPO G1A1 GOPO GOPO G1A1 GOPO GOPO G1A1 GOPO GOPO GOPO GOPO G2A2 GOPO GOPO G1P1 G1A1 GOPO G1A1 GOPO GOPO GOPO GOPO GOPO GOPO GOPO GOPO GOPO

3 4 4 9 7 2 2 2.5 2 5 2.5 10 3 5 2 2 3 3 4 5 5 8 2 4 2 3 6 3 3 4 2 4

12 8 3 7 4 15 6 10 3 16 7 4 12 5 5 6 4 4 5 5

9 6 2 6 4 10 5 7 2 9 3 3 8 3 4 3 4 3 3 4 5 9 10 4 6 5 11 2 4 5 4 5 3 6 7 19

4 4 2 4 4 4 5 4 2 4 3 3 4 3 4 3 4 3 3 4 5 4 4 4 4 5 4 2 4 5 4 5 3 4 4 4

8

3 5 4

8

16 14 6 8 7 13 4 7 8 7 7 5 11 12 23

*G, Gravida; P, para; A, spontaneous abortion.

whether it is s 0.05. By contrast, this correction is not needed to answer the question of whether there is increased sharing of three or more of the HLA-A, B, DR, DQ antigens or two or more of the HLA-B, DR, DQ or HLA-A, B, DR antigens, because only one factor is being considered in this comparison. This analysis strengthens the argument that the sharing of a specific HLA antigen is not important, whereas the sharing of groups of antigens as markers for other genes in a specific segment of the HLA region is critical.

Comment The data from couples having recurrent spontaneous abortions, J3 gestational trophoblastic tumors,12 and failures in NF show that they all share HLA antigens more frequently than normally fertile couples (fable VII). The maximal level of sharing is in the BDRDQ region, . and no specific antigen is present at a higher frequency than normal at any particular locus. These findings indicate that a gene(s) or genetic defect (we favor the latter on the basis of studies in experimental animals")

in the BDRDQ region is the cause of these diseases. This conclusion is supported by epidemiologic studies showing a higher prevalence of recurrent spontaneous abortion, cancer, and congenital anomalies in the families of couples experiencing recurrent spontaneous abortions or gestational trophoblastic tumors. 15 A corollary line of supportive evidence comes from the failure of immunotherapy to influence the prevalence of recurrent spontaneous abortions in the population studied,14 and this finding is consistent with a genetic basis for the fundamental reproductive defect. Further support for the genetic hypothesis for the cause of these reproductive defects now comes from the comparative genetics of the major histocompatibility complex. At the time that the association between sharing two or more of the HLA-A, B, DR loci and recurrent spontaneous abortions was proposed,16. 17 studies by two groups of investigators showed that the genes in the central portion of the major histocompatibility complex are in tight linkage disequilibrium and function en bloc. Egea et aJ.24 identified this block as a

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Volume 170, Number 1, Part 1 Am J Obstet Gynecol

67

Table III. HLA typing of infertile couples in whom the woman successfully underwent IVF and tubal embryo transfer Antigen Patient

Sex

51

F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M

52 53 54 55 56 57 58 59 510 5II 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534

A

II,33 2 2, 33 II II,24 II,33 2 24 II,33 2, II 2 II II,3I 31,33 24 II,24 2 II,24 2, II 2, 24 2, II II, 24 2, II II,26 2, II 24 2, II 2,25 II 2, II 2, 33 2, 31 II II,24 2, II II,33 2, 24 24, 26 2,33 2 24 2, 30 I, 2 I, II 24 31 II 2 26, 33 II,24 2 2, II II,24 2, 31 24 2, II 2 II, 33 II 33 2, 33 II 2, II II,24 24 II 2 II

I

B

58,75, w4, w6 61,70, w6 46, 58, w4, w6 46,60, w6 46,62, w6 58,60,w4,w6 58, 60, w4, w6 39,56, w6 58,60,w4,w6 13,60, w4, w6 55, w6 55,62, w6 13,61, w4, w6 54, w4, w6 60, w6 35,39, w6 38,58,w4 13, w4, w6 46,60,w6 38, 46, w4, w6 60, w6 54,61, w6 60, w6 54,61, w6 13, 70, w4, w6 13,60, w4, w6 46,60, w6 62, w6 I3,5I,w4 60,62, w6 55, 58, w4, w6 38,51, w4 46,52,w4,w6 55,60, w6 46,54, w6 58, 61, w4, w6 46,70, w6 60, w6 54,58, w4 46,52, w4 27, 38, w4, w6 8,75,w6 51, w4 35, 53, w4, w6 58,60, w6 13, 60, w4, w6 60,62,w6 46 39, 58, w4, w6 46,60,w6 44, 60, w4, w6 46,51, w4 35,75, w4, w6 13, 54, w4, w6 44,51 27 46 58, 60, w4, w6 51,60, w4, w6 13,39, w6 48, 58, w4, w6 39,62, w6 60,46, w6 60, w4, w6 58, 60, w4, w6 54,62, w6 35,46, w6 60,75, w6

I

c w3 w7 wII w4, wII wII w3 w3 wI, w7 w3, w7 w3, w7 wI wI, w4 w6,w7 wI, w7 w3 w3 w3,w7 wI, w3 w7, wII w3,wII w3,w7 wI, w7 w7 wI, w7 w3, w7 w6,w7 w7, wII w3 w3 w3 w7, wII w7 wII wI, w7 wII w3 wII w7 wI wII w7 w7 wI,w7 w4 w3, w7 w6,w7 w4, wlO wlO w7, wIO w9, wII w7 wII wI, wlO wI wII w7, wlO w3,w7 w4 wII w9 wIO wI, w4 w4, wII

Sharing

I

DR

4, 12, w52 14, 15, w52 3, 4, w52, w53 4,9, w53 9, 12, w52 14, 16, w52 2,3, w52 4, w52, w53 3,5, w52 2, w52 15, w52 4, w53 5, 12, w52 4,9, w53 9, 15, w53 4,9, w53 13, 16, w52 9, 16, w53 7,9, w53 9, 16, w53 15 4, w52 8, II w52 4 II, 15, w52 7, w53 4,9, w53 4, 15 9, II, w52, w53 9, w53 8, 14, w52 9, 16, w53 9, 15, w53 12, 15, w52 4, II, w52, w53 14, w52 15 9, 15, w53 3, 14, w52 9,w53 4, w53 3,8, w52 15 12, 13, w52 8, 14, w52 4, 7, w53 4, II, w52, w53 4, w53 4, 14, w52, w53 9, 12, w52, w53 7, 11, w52, w53 4,9, w53 8, 12, w52 4, 16, w53 II, w52, w53 8, II, w52 9, w53 12, 17, w52 8, 16, w52 4, 17, w52, w53 I2,I7,w52 II, 15, w52 4, 12, w53 4, 15, w53 II,I7,w52 4, w53 8, II, w52 8,12, w52

I

DQ

ABDRDQ

w7 w6 w7 w7 w3 w3,w6 wI, w2 wI, w3 w2,w7 wI w3,w5 w3 w7

°

w3,w6 w3 w6 w3,w6 w2,w3 w3,w6 wI wI w5,w7

3

w5,w7 w2 w5 w3,w7 w3 w4,w6 w3,w6 w5 w5 w7 w6 w6 w6 w2, w6 w5 w3 wI w6 w2,w6 w5 w2,w4 w7 w5 w3, w6 w3 w2,w7 w3 w2,w4 w3,w5 w6, w7 w6,w7 w3 w3 w6 w3 w2, w7 w5,w7 w4,w7 w3,w5 w2,w7 w3 wI, w7 w4,w7

3

I

BDRDQ

° 3

I ABDR

°

2

2

°

2 2

2

2

° 2

2

2

4

3

2

3

2

° 2

2

2

3

2

2

2

2

2 3

3

° 2

° 2

2

° °

° °

° °

°

°

°

3

2

° ° °

° 2

° 2

2

°

2

3

°

68

He et al.

January 1994 Am J Obstet Gynecol

Table IV. HLA typing of infertile couples in whom the woman failed to conceive after IVF and tubal embryo transfer Antigen Patient

Fl F2 F3 F4 F5 F6 F7 F8 F9 FlO F11 Fl2 Fl3 Fl4 F15 F16 Fl7 F18 F19 F20 F21 F22 F23 F24 F25 F26 F27 F28 F29 F30 F31 F32 F33

Sex

F

M

F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M F M

A

24 2, 11 11,26 2,31 11,24 2 2,24 2, 11 2, 11 2, 11 2, 24 31 2, 24 2, 11 2,11 11,31 11,24 2,24 11,24 11 11 2, 11 24,33 11 11,24 2,11 2, 11 11,24 11,30 2, 11 11,24 2,24 11,30 11 2, 11 2, 11 2, 11 2,11 2, 11 2, 24 2, 11 2,11 11,24 11 11,24 11,24 11 11 24, 33 2, 33 11 24 24, 26 2,11 11,24 24, 31 1, 2 11 2, 24 2, 11 11 2, 24 11,24 2, 34 11,24 2,11

I

B

13, 46, w4, w6 39,55,w6 35, 58, w4, w6 54,70, w6 46,60, w6 60,62, w6 37, 46, w4, w6 13, 60, w4, w6 60, w6 46,60, w6 60, w6 53,58, w4 38,51, w4, w6 35,51, w4, w6 62, w6 13,57, w4 60, w6, w4 38,60,w4,w6 54,55, w6 13,46, w6 13,46, w4, w6 54,60, w6 58, 60, w4, w6 13,60, w4, w6 13, 60, w4, w6 39,46, w6 13, 55, w4, w6 51,55, w4, w6 13,60, w4, w6 46,70, w6 27,46,w4 60, w6 13,48, w4, w6 39,75, w6 60, w6 13,38, w4, w6 60,62, w6 27,38, w4, w6 27,53, w4, w6 60, w6 17, 62, w4, w6 38, 62, w4, w6 39, 52, w4, w6 13, 38, w4, w6 38, 60, w4, w6 51,75, w4, w6 58,60,w4,w6 58,75, w4, w6 38,46,w4 38,58, w4 27,62, w4, w6 46,60, w6 54,60, w6 52, 75, w4, w6 60,75, w6 51,60, w4, w6 37,46,w4 39,55, w6 46,60, w6 46,60, w6 58, 60, w4, w6 46,52,w4 39,60,w6 46,75, w6 46,60, w6 48,60, w6

I

c w6, wll wI, w7 w3,w4 wl,w4 w11 w3 w6, w11 w3,w6 w7 w7, w11 w7 w3 wI, w3 w6 w3,w7 w7 wi wI w3 wI w7, wl0 w6,w7 w6, w11 wI, wl0 wI w9 w11 w11 w4,w7 w9 w7, wlO w7, wlO w7,w9 w7 w9 w9 wlO wlO wlO w11 wlO wI w11 wI, w7 wl0 wl0 w6, w11 wI wI w7, w11 w9 w7, w11 wl0 w6, w11 w11 w7

Sharing

I

DR

4, 7, w53 12, 14, w52 3, w52 4, w53 15, w52 4, 15 4,9, w53 12, w52, w53 8, 15, w52 4, 8, w52, w53 12, 15, w52 12, w52 12, w52 15 4, w53 7, w53 4, 14, w52, w53 4,9, w53 12, 14, w52 14, 15, w52 9,16,w53 4, w53 4,8, w52 9, w52, w53 7, 15, w53 9,15, w53 4, 16, w53 9, 14, w52, w53 12, 16, w53 12,16, w52 9, 12, w52, w53 4,9, w53 6, 9, w52, w53 4, 12, w52, w53 9,16,w53 4, w52, w53 3,12, w52 7, 14, w52, w53 9, 17, w52, w53 15 12, 17, w52 12, 15, w52 4,9, w53 14, 15, w52 9,15, w53 12, 15, w52 11, 14, w52 15 8, w52 4, 12, w52, w53 11, 12, w52 9, 11, w52, w53 4, w53 8, 12, w52 12, 15, w52 11, 14, w52 10, 16 4, w53 16 9,14,w52,w53 14, w52 8, 12, w52 8, 12, w52 12, 15, w52 9, 15, w53 11, w52

I

DQ

w2,w7 w5,w7 w2 w7 w5 w5 w3,w5 w3,w5 w6 w3,w6 w5,w7 w7 w6 w6 w3 w2, w3 w7 w7 w5 w5 w5 w3,w6 w3,w6 w2,w6 w6 w5 w5 w6,w7 w6,w7 w3 w3 w7 w6 wi, w6 w2,w7 w6 w3 w5 w7 w6,w7 w3 w5 w3,w6 w6,w7 w6,w7 w6 w7 w7 w7 w7 w3 w3 w6,w7 w5,w7 w5 w3,w5 w5 w5 w6 w6,w7 w6,w7 w6,w7 w3,w6 w5,w7

ABDRDQ

° 3

I BDRDQ I ABDR

°

3

° ° 2

2 4

3

2

2

3

2

2

4

3

3

3

2

2

3

2

2

° 2

3

2

2

3

2

2

2

2

3

2

2

4

3

2

° ° ° 3

°

3

2

2

3

2

2

3

2

2

2

2

3

2

3

2

2

2

2

° 2

° 2

° 2

Ho et al.

Volume 170, Number 1, Part 1 Am] Obstet Gynecol

69

Table IV-Cont'd Antigen Patient F34 F35 F36

A

Sex F

M

F

M

F

M

I

I

B 58,w4,w6 35,75,w6 46,60,w6 54,60,w6 46,58,w4 46, 51, w4, w6

24 11,24 2 11,24 2 2,11

Sharing

I

c w9 w9 w11 wI w11 wI

I

DR 12,13, w52 12,15, w52 9, w53 12,16, w52 4,13,w52 4,9, w53

DQ

ABDRDQ

w6,w7 w6,w7 w3 w3,w5 w3,w5 w3

I BDRDQ I ABDR

3

2

2

2

4

3

2

3

Table V. HLA typing and obstetric history of infertile couples who successfully underwent IVF and tubal embryo transfer but aborted in the first trimester

Patient

Obstetric history*

Al

GIPI

A2

GOPO

A3

GOPO

A4

G2A2

A5

GOPO

A6

GOPO

Antigen Sex F

M

F

M

F

M

F

M

F

M

F

M

A 2,31 2, 11 2,11 11 24 11,29 11 2,11 2,11 1,2 11 2,11

I

I

B 37, 75, w4, w6 51,62,w4,w6 13, 55, w4, w6 51, 55,w4,w6 46,60,w6 7,27,w4,w6 62, w6 46,w6 35, 53, w4, w6 51, w4, w6 58, 60, w4, w6 22, 48,w4,w6

C

Sharing

I

w7 w4 wI wI w11 w9 w9, w11 w4 wl,w7 wl0 wl0

DR

I

12, w52 4,9, w53 4,16,w53 4, 12, w52, w53 9, 12, w52, w53 10, w52 9, w53 9, w53 w12, w13, w52 w15 15, 17, w52 4, 17, w52, w53

DQ

ABDRDQ

wl,w3 w3 w5 w5 w7 w6 w3 w3 w2,w6 w6 w2,w6 w2,w7

2

l BDRDQ IABDR

4

3

3

0

0

0

3

2

2

2

2

2 3

*G, Gravida; P, para; A, spontaneous abortion.

Table VI. The effect of HLA sharing on the success of IVF and tubal embryo transfer Sharing of RIA loci

Success with tubal embryo transfer (n = 34) Age (yr, mean ± SE 33.3 ± 4.4 (22-41) and range) 3.9 ± 1.7 (2-9) Duration of infertility (yr, mean ± SE and range) 71.1 ± 20.8 Fertilization rate (%) 40.6 ± 20.1 Implantation rate (%) HLA loci (No.) 17 (50.0%) A 6 (17.6%) B 15 (44.1%) C 12 (35.3%) DR 18 (52.9%) DQ 6 (17.6%) ABDRDQ 2: 3 11 (32.4%) BDRDQ 2: 2 9 (26.5%) ABDR 2: 2

Significancet

Failure with tubal embryo transfer (n = 36)*

Normally fertile controls (n = 51)

33.4 ± 3.9 (26-42)

32.3 ± 3.4 (28-38)

Success vs failure with IVF-tubal embryo transfer

Normal controls vs failure with IVF-tubal embryo transfer

Success with IVF-tubal embryo transfer vs normal controls

4.0 ± 2.2 (2-10) 69.0 ± 13.1

24 14 12 14 29 16 22 17

(66.7%) (38.9%) (33.3%) (38.9%) (80.5%) (44.4%) (61.1%) (47.2%)

28 14 18 18 29 11 16 19

(54.9%) (27.4%) (35.3%) (35.3%) (56.8%) (21.5%) (31.4%) (37.2%)

P= P= P= P= P= P= P= P=

0.12 0.044 0.25 0.47 0.013 0.015 0.015 0.060

P= P= P= P= P= P= P= P=

0.19 0.19 0.52 0.45 0.D18 0.021 0.006 0.24

P= P= P= P= P= P= P= P=

0.41 0.22 0.28 0.41 0.45 0.44 0.55 0.21

*An additional six women had recognized spontaneous abortions. If they are added to the failure group (n = 42), the results of the analysis remain the same. tFisher exact test (one-tailed). If the two-tailed Fisher exact test was used, all significant differences by the one-tailed test in the sharing of three or four HLA antigens would be significant at p S 0.034.

70

He et al.

January 1994 Am J Obstet Gynecol

ll-OH, C lo Br, HIP7O, TNF, cia. I

GJo-l

I

:

,

r-- -- .... -_ .. --_ ..... -- "-"'7" .... -

~

:

t.

..... - - - - _ .... - - - - - - -,

I

.. ------ ... -------------- ...... ---------- .. -... ----- .. -- .. ----- ......... --~

Fig. 1. Comparison of major histocompatibility complexes of human, mouse, and rat; approximate size of each major histocompatibility complex region is also given. Solid circles, centromere; boxes with solid margins, classical class I loci; boxes with dashed margins, nonclassical class I loci; circles, class II loci; TAp, loci encoding class I antigen transporter proteins; Glo-I, locus encoding enzyme glyoxylase-l. Class III loci are 2 I-hydroxylase (2I-OH), fourth component of complement (C 4 ), properdin factor B (Bj), second component of complement (C2 ), heat shock protein 70 (HsP70), and tumor necrosis factor (TNF) (a and 13 loci). Genes affecting growth and development (stippled areas) are Ped (preimplantation embryonic development) in mouse (genes affecting lung development are also in this region) and rcc (resistance to chemical carcinogens),ft (fertility) and dw-3 (body size), all of which constitute grc in rat. There is translocation in order of loci in mouse and rat major histocompatibility complexes relative to that in all other species, including human, so that class II and class III loci are located between major class I loci (K and D in mouse and A and E in rat). The way in which this translocation in mouse and rat occurred is shown by dashed lines under human major histocompatibility complex; it shows homology of class II and class III loci in all three species.

Table VII. Comparison of HLA sharing in couples having recurrent spontaneous abortions, gestational trophoblastic tumors, and failing IVF and tubal embryo transfer compared with normally fertile couples HLA sharing (significance)*, t

HLA locus

Recurrent spontaneous abortion (n = I23)t

ABDRDQ ~ 3 BDRDQ ~ 2 ABDR ~ 2

p = 0.004

P = 0.006 P = 0.053

Gestational trophoblastic tumor (n = 26):

p = 0.012

P= P=

0.002 0.126

Failure of IVF-tubal embryo transfer (n = 36):

p = 0.021 P = 0.006 P = 0.239

*References 13, 14, and this article. tFisher exact test (one-tailed). tNumber of couples.

"complotype" that spanned the Bf-C2-C4A-C4B genes and later expanded this region to include the HLA-B and HLA-DR genes, to form an "extended haplotype." Degli-Esposti et al. 25 showed that the region from HLAB to HLA-DQ functioned as a unit, on the basis of extensive population studies, and they referred to these units as "ancestral haplotypes." Approximately 40 ancestral haplotypes or recombinants among them were adequate to explain all the major histocompatibility complex variation in the white population that they

studied; similar findings were observed in Asian and black populations. Both groups showed that better correlations with disease susceptibility occurred with these extended m~or histocompatibility complex units than with a single major histocompatibility complex locus 24 • 26 : insulin-dependent diabetes mellitus, myasthenia gravis, asthma and rhinitis, congenital adrenal hyperplasia (because of a deletion of the Cyp21 [21-0H] locus), gluten-sensitive enteropathy, pemphigus vulgaris, and ulcerative colitis. They ascribed this associa-

Volume 170, Number 1, Part 1 Am J Obstet Gynecol

tion to major histocompatibility complex or m~or histocompatibility complex-linked genes other than the class I and class II genes. Similarly, we have identified genes influencing growth, reproduction, and resistance to chemical carcinogens in the m~or histocompatibility complexlinked region of the rat that are not class I or class II genes. 1. 10, 11, 18 The position of these genes in the rat, and similar genes in the mouse, map to a homologous position in the human (Fig. 1). The organization of the major histocompatibility complex in all species except the rat and the mouse is the same as in the human. 18 In the rat and mouse there appears to have been a translocation so that the class II and class III regions were translocated between the two major class I regions. Considering this translocation, the BDRDQ region in the human, which affects implantation, developmental defects, and the development of cancer/ 8 is homologous to the gre region in the rat, which affects growth, development, and resistance to chemical carcinogens, and to the Ped locus in the mouse, which affect preimplantation embryonic development. Therefore quite similar genes in all three species map to the homologous position, and this observation supports the generality of their evolutionary importance. Their explicit identification will provide greater insight into the reproductive process and will provide diagnostic approaches to predicting reproductive outcome. At the current time we consider that sharing two or three of the HLA-B, DR, DQ antigens between partners is a factor leading to an unsuccessful reproductive outcome after natural mating or IVF. REFERENCES 1. Gill T] Ill. MHC-linked genes affecting reproduction, development, and susceptibility to cancer. In: Coulam C, Faulk WP, McIntyre ]A, eds. Immunological obstetrics. New York: Norton, 1992:103-12. 2. Coulam CB, Moore SB, O'Fallon W, Investigating unexplained infertility. AM] OBSTET GYNECOL 1988;158:137481. 3. Crosignani PG, Walters DE, Soliani A. The ESHRE multicentre trial on the treatment of unexplained infertility: a preliminary report. European Society of Human Reproduction and Embryology. Hum Reprod 1991;6:953-8. 4. Yang YS, Ho HN, Lien YR, Hsieh CY, Lee IT. Treatment of couples witb longstanding unexplained infertility: gamete intrafallopian transfer (GIFT) versus tubal embryo transfer (TET). ] Formosan Med Assoc 1991;90:547-50. 5. Yang YS, Hwang ]L, Ho HN, et al. Translaparoscopic tubal embryo transfer-preliminary experience at National Taiwan University Hospital. Asian Oceanic] Obstet GynecoI1991;17:255-9. 6. Hwang ]L, Yang YS, Ho HN, et al. Translaparoscopic tubal embryo transfer as a treatment of unexplained infertility.] Obstet Gynecol ROC 1991;30:198-202. 7. Gurgan T, Kisnisci HA, Varali H, et al. Evaluation of the functional status of the fallopian tubes in unexplained infertility with radionuclide hysterosalpingography. Gynecol Obstet Invest 1991;32:224-6.

Ho at al.

71

8. Murdoch AP, Harris M, Mahroo M, William M, Dunlop W. Gamete intrafallopian transfer (GIFT) compared with intrauterine insemination in the treatment of unexplained infertility. Br] Obstet GynaecoI1991;98:1107-11. 9. Mackenna AI, Zegers-Hochschild F, Fernandez EO, et al. Fertilization rate in couples witb unexplained infertility. Hum Reprod 1992;7:223-6. 10. Kunz HW, Gill T] III, Dixon im; Taylor FH, Greiner DL. The growth and reproduction complex in the rat: genes linked. to tbe major histocompatibility complex which affect development.] Exp Med 1980;152:1506-18. 11. Melhem MF; Kunz HW, Gill T] III. An MHC-linked locus in the rat critically influences resistance to DEN carcinogenesis. Proc Nat! Acad Sci USA 1993;90:1967-71. 12. Ho HN, Gill T] III, Klionsky B, et aI. Difference between white and Chinese populations in HLA sharing and gestational trophoblastic tumors. AM] OBSTET GYNECOL 1989; 88:474-7. 13. Ho HN, Gill T] III, Hsieh RP, Hsieh F], Lee IT. Sharing of human .leukocyte antigens (HLA) in primary and secondary recurrent spontaneous abortions. AM ] OBSTET GYNECOL 1990;163:178-88. 14. Ho HN,. Gill T] III, Hsieh F], Jiang ]], Lee IT, Hsieh CY. Immunotherapy for recurrent spontaneous abortion in a Chinese population. Am ] Reprod Immunol 1991;25: 10-5. 15. Ho HN, Gill T] III, Hsieh CY, Yang YS, Lee IT. The prevalence of recurrent spontaneous abortions, cancer, and congenital anomalies in the families of couples having recurrent spontaneous abortions or gestational trophoblastic tumors. AM] OBSTET GYNECOL 1991;165:461-5. 16. Gill T] III. Immunogenetics of spontaneous abortions in humans. Transplantation 1983;35: 1-6. 17. Gill T] III. The borderland of embryogenesis and carcinogenesis: MHC-linked genes affecting development and tbeir possible relationship to the development of cancer. BBA Cancer Rev 1984;738:93-102. 18. Gill T] Ill. Reproductive immunology and immunogenetics. In: Knobil E, Neill ]D, eds. The physiology of reproduction. 2nd ed. New York: Raven Press [In press]. 19. Yang YS, Ho HN, Lien YR, et al. The use of a longacting gonadotropin-releasing hormone analog (D-Trp-6LHRH) for improvement of ovarian stimulation in assisted conception programs. ] Formosan Med Assoc 1991;90: 1081-5. 20. Quinn PC, Kerin ]F, Warnes GM. Improved pregnancy rate in human in vitro fertilization witb tbe use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8. 21. Lopata A, Patullo M], Chang A, James B. A metbod for collecting motile spermatozoa from human semen. Fertil Steril 1976;27:677-84. 22. Snedecor GW, Cochran WG. Statistical methods. Ames: Iowa State University, 1989:116. 23. Vardimon D, Locker ], Kunz HW, Gill T] III. Physical mapping of the MHC and grc by pulse field electrophoresis. Immunogenetics 1992;35:166-75. 24. Egea GE, Yunis I, Spies T, et al. Associations of polymorphisms in the HLA-B region with extended haplotypes. Immunogenetics 1991 ;33:4-11. 25. Degli-Esposti MA, Leaver AL, Christiansen FT, Witt CS, Abraham L], Dawkins RL. Ancestral haplotypes: conserved population MHC haplotypes. Hum Immunol 1992; 34:242-52. 26. Tokunaga K, Saueracker G, Kay PH, Christiansen FT, Anand R, Dawkins RL. Extensive deletions and insertions in different MHC supratype~ detected by pulsed field gel electrophoresis.] Exp Med 1988;168:933-40.