EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1977,7/l, 0 Elsevier/North-Holland Biomedical Press
l-4
The transport of chorionic gonadotropin through the reproductive tract* B.B. Saxena, S. Kaali and R. Landesman Cornell University Medical College and Jewish Memorial Hospital, New York, N. Y., U.S.A.
SAXENA, B.B., KAALI, S. and LANDESMAN, R. (1977): The transport of chorionic gonadotropin through the reproductive tract. Europ. J. Obstet. Gynec. reprod Biol., 7/l, l-4. In order to demonstrate the ability of HCG to cross the uterine wall prior to placental vascularization, inert implants impregnated with HCG were placed into the uterine cavity of the rabbit. HCG was able to pass through the uterine wall into the circulation in a dose-response relationship and was detected in the blood within 30 min. In the human, HCG applied topically on postovulatory endometrium could be detected in the peripheral circulation within 6 h. These results indicate that HCG molecules can cross the reproductive tract in the rabbit and human without vascular connections. uterine wall; rabbits; human; postovulatory
endometrium
face of intact mouse embryo prior to implantation. Haour, Charming, and Saxena (1975) have further shown that blastocyst fluid from rabbits prior to implantation is capable of inducing luteinization and progesterone synthesis in pig and monkey granulosa cells in in vitro cultures. Recently, Dickman and Dey (1974) have shown evidence for the presence of dehydrogenases and synthesis of 3/3-hydrosteroid, estradiol and progesterone, in the blastocysts of mouse, rat, hamster and rabbit prior to implantation, and have suggested the possibility for the existence of luteotropic signal from the blastocyst. One report has failed to find luteotropic effect of blastocyst fluid of rabbits (Sundarum, Connell and Passantino, 1975). Although the chemical nature and source of the luteotropic material has not been established, these observations have raised the question as to the transport of the luteotropic material prior to implantation. We describe here experiments to show that exogenous HCG passes the reproductive tract of rabbit and human without placental vascularization.
Introduction
The detection of HCG as early as day 6 and 7 after conception by radioimmuno (Kosasa, Levesque, Taymor and Goldstein, 1974) and radioreceptorassay (Saxena, Hasan, Haour and Schmidt-Gollwitzer, 1974) in humans suggests that the luteotropic material prior to or at the initiation of implantation may provide an early maternal recognition of the fetus and sustain the corpus luteum of pregnancy. Further evidence is provided by the studies of Fujimoto, Euker, Riegel and Dukelow (1975) by radioimmunoassay, and Haour and Saxena (1974) by radioreceptorassay of HCG-LH, who have demonstrated the presence of luteotropic material in the rabbit blastocyst fluid prior to implantation. Wiley (1974) using immunofluorescence, has found HCG-like material on the sur-
* Supported by Grants from the Rockefeller Foundation (GA-HS-7406) and the National Institutes of Health Grant (HD-09004) from National Institutes of Health and World Health Organization. 1
2 Material and methods Studies on rabbits
Samples of HCG containing 12,000 IU/mg were provided by Dr. R.E. Canfield and Dr. O.P. Bahl. Aliquots of 10,100 and 200 pg of HCG in saline were impregnated into 1 mm X 1 cm implants provided by Dr. G. Gupta (Population Council, Rockefeller University, New York, N.Y). The HCG implants were inserted in the uterine horn of rabbits under aseptic conditions in the following manner. Rabbits were anesthesized with nembutal. A ventral incision was made through the vaginal wall. A 2-inch Tygon tube of 2 mm diameter was inserted into the uterus and the implant was then pushed into the uterine cavity via the indwelling Tygon tube. Care was taken to avoid any laceration of tissue around the cervix and inside the uterus. Sham operation was performed using implants without HCG. The blood was collected at 15and30minandat 1,2,and4handonthefollowing 3 days. On day 4, the animals were sacrificed; the implants were recovered from the uterus and the ovaries were removed. All serum samples were analyzed for HCG by the radioreceptorassay using plasma membrane of bovine corpora lutea of pregnancy and HCG labeled with 12’I by lactoperoxidase as described previously (Saxena et al., 1974). The blank, control and standards were made in serum containing basal levels of LH of equal protein concentration in the standard and unknown and to measure increase in luteotropic material over the basal levels. For example, 50 /J of rabbit serum, and 100/J of human serum were added to respective standards.
B.B.Saxena et al.: HCG transport through the reproductive tract
placed in the distal tubes and in the other in the more proximal portion close to the body of the uterus. In 2 final patients, HCG was deposited around the ovaries in the peritoneal cavity. Blood samples were obtained prior to and at 15 and 30 min and 1,2,4,8, 16, 24, and 48 h after the administration of HCG. The serum samples were analyzed for HCG by the radioreceptorassay.
Results and Discussion
The peripheral levels of HCG observed after implantation of 10, 100 and 200 pg in one uterine horn of the rabbit are shown in Figure 1. The HCG appeared in the blood as early as 15 min after implantation. From the three implants, HCG levels as high as 90, 165, and 625 ng/ml, respectively, were observed in the blood within 30 min to 2 h. On day 3, the blood levels were still elevated in the rabbits car-
b-ice ; NC/Ml
Studies in women
In one subject 5,000 IU of HCG was injected intramuscularly into the lateral buttock. An additional 7 women each received 5,000 IU of HCG in 2 ml of normal saline into various areas of the reproductive tract. In 3, HCG was instilled into the uterine cavity via a cervical cannula, after the tubes were tied at the uterotubal junction. The cannula remained in place for 1 h to maintain the HCG in contact with the endometrial surfaces. In 2, the HCG was instilled into the tubal lumen: 1 ml in the right and the second ml in the left. In 1 of these 2 patients, the HCG was
10
5
1
IMPLANT
TIME
24 ;
48
72
HOUR
Fig. 1. Detection of HCG in serum by radioreceptorassay following intrauterine implantation of HCG in rabbits. Sharnoperated rabbits showed no HCG in the serum.
3
B.B. Saxena et al.: HCG transport through the reproductive tract
rying implants of 100 and 200 pugHCG. The presence of up to 20-25 corpora lutea in the ovary (Fig. 2) demonstrated the HCG released by the implants crossed the uterine wall into the peripheral circulation and luteinized the ovaries. The sham-operated rabbits showed neither any HCG in the circulation nor stimulation of corpora lutea. When 5,000 IU HCG were injected intramuscularly, the highest plasma concentration occurred at 2 h (top channel; Fig. 3). Transport of HCG from the pelvic peritoneum in the ovarian region on the 6th and 8th days of the cycle was also detected at 8 h reaching a maximum between 16 and 24 h. After instillation of HCG on days 14 and 16, HCG was sustained up to a 48-h period in the blood. However, HCG was not detected in blood for up to 48 h when applied to the endosalpinx on days 10 and 26 of the cycle or to the endometrium on day 5. These results
Fig. 2. Superovulated ovary recovered 3 days following intrauterine implantation of 200 fig HCG in rabbit (left). The ovary from the sham-operated control rabbit is shown on the right.
I
I
ovary
1 ‘IIt
Day
6
Endometrium
Day
14
Endometrium
Day
15
,I Endometrium
10 I,;
Endosalpinx
0
-0
.l
Day Day
5 10 6 26
I
A
1 Hours
2 post
hCG
I
4
1
8
16
24
48
administration
Fig. 3. The detection of HCG in the serum following introduction of HCG into the endometrium, cavities as compared to intramuscular injection with 5,000 IU of HCG, in women.
endosalpinx,
and peritoneal
4
suggest that in the event a preimplantation HCG-like material is elaborated, it can cross the uterine cavities and reach the peripheral circulation. Methods to inactivate luteotropins by antibodies in early pregnancy are being considered as targets for contraception @Learn and Lunn, 1975; Stevens, 1973; Stevens and Crystle, 1973; Bahl, Pandyan and Moyle, 1975; Segal, 1976).
References Bahl, O.P., Pandyan, P.R., Moyle, W.R. and Kobayashi, Y. (1975): Chorionic gonadotropin in fertility and reproduction. In: Advances in Fertility Regulation through Basic Research. Editors: W.A. Sadler and S. Segal. Plenum Press, New York. Dickman, Z. and Dey, S.K. (1974): Evidence that A5-3p-hydroxysteroid dehydrogenase activity in rat blastocyst is autonomous.J. Endocr., 61, 513. Fujimoto, S., Euker, J.S., Riegel, G.D. and Dukelow, W.R. (1975): On a substance crossreacting with luteinizing hormone in the preimplantation blastocyst fluid of the rabbit. Proc. Japan Acad., 51, 123. Haour, F., Saxena, B.B. (1974): Detection of agonadotropin in rabbit blastocyst before implantation. Science, 185, 444. Haour, F., Charming, C. and Saxena, B.B. (1975): A stimula-
B.B. Saxena et al.: HCG transport through the reproductive tract tory effect of prelmplantation rabbit blastocyst fluid upon luteinization of monkey granulosa cell cultures. In: Abstracts, Endocrine Society 57th Annual Meeting, p. 107. Hearn, J.P. and Lunn, S.F. (1975): The endocrinology of the ovarian cycle and of pregnancy in the Maroset monkeys. J. Endocr., 65, 1. Kosasa, T.S., Levesque, L.A., Taymor, M.L. and Goldstein, D.P. (1974): Measurement of early chorionic activity with a radioimmunoassay specific for human chorionic gonadotropin following spontaneous and induced ovulation. Fertil. and Steril., 2.5, 2 11. Saxena, B.B., Hasan, S.H., Haour, F. and Schmidt-Gollwitzer, M. (1974): Radioreceptorassay of human chorionic gonadotropin: Detection of early pregnancy. Science 184, 793. Segal, S.J. (1976): Immunological methods to prevent pregnancy - an editorial comment. Contraception, 13, 125. Stevens, V.C. (1973): Immunization of female baboons with hapten coupled gonadotropins. Obstet. and Gynec., 42, 495. Stevens, V.C. and Crystle, C.D. (1973): Effects of immunization with hapten coupled hCG on the human menstrual cycle. Obstet. and Gynec., 42, 485. Sundaram, K., Connell, K.G., Passantino, T. (1975): Control of corpus Iuteum function in the pregnant rabbit: Absence of hCGlike material in the blastocyst. In: Abstracts,Endocrine Society 57th Annual Meeting, p. 107. Wiley, L.D. (1974): Presence of a gonadotropin on the surface of preimplanted mouse embryos. Nature (London), 252, 715.
l-4
The transport of chorionic gonadotropin through the reproductive tract* B.B. Saxena, S. Kaali and R. Landesman Cornell University Medical College and Jewish Memorial Hospital, New York, N. Y., U.S.A.
SAXENA, B.B., KAALI, S. and LANDESMAN, R. (1977): The transport of chorionic gonadotropin through the reproductive tract. Europ. J. Obstet. Gynec. reprod Biol., 7/l, l-4. In order to demonstrate the ability of HCG to cross the uterine wall prior to placental vascularization, inert implants impregnated with HCG were placed into the uterine cavity of the rabbit. HCG was able to pass through the uterine wall into the circulation in a dose-response relationship and was detected in the blood within 30 min. In the human, HCG applied topically on postovulatory endometrium could be detected in the peripheral circulation within 6 h. These results indicate that HCG molecules can cross the reproductive tract in the rabbit and human without vascular connections. uterine wall; rabbits; human; postovulatory
endometrium
face of intact mouse embryo prior to implantation. Haour, Charming, and Saxena (1975) have further shown that blastocyst fluid from rabbits prior to implantation is capable of inducing luteinization and progesterone synthesis in pig and monkey granulosa cells in in vitro cultures. Recently, Dickman and Dey (1974) have shown evidence for the presence of dehydrogenases and synthesis of 3/3-hydrosteroid, estradiol and progesterone, in the blastocysts of mouse, rat, hamster and rabbit prior to implantation, and have suggested the possibility for the existence of luteotropic signal from the blastocyst. One report has failed to find luteotropic effect of blastocyst fluid of rabbits (Sundarum, Connell and Passantino, 1975). Although the chemical nature and source of the luteotropic material has not been established, these observations have raised the question as to the transport of the luteotropic material prior to implantation. We describe here experiments to show that exogenous HCG passes the reproductive tract of rabbit and human without placental vascularization.
Introduction
The detection of HCG as early as day 6 and 7 after conception by radioimmuno (Kosasa, Levesque, Taymor and Goldstein, 1974) and radioreceptorassay (Saxena, Hasan, Haour and Schmidt-Gollwitzer, 1974) in humans suggests that the luteotropic material prior to or at the initiation of implantation may provide an early maternal recognition of the fetus and sustain the corpus luteum of pregnancy. Further evidence is provided by the studies of Fujimoto, Euker, Riegel and Dukelow (1975) by radioimmunoassay, and Haour and Saxena (1974) by radioreceptorassay of HCG-LH, who have demonstrated the presence of luteotropic material in the rabbit blastocyst fluid prior to implantation. Wiley (1974) using immunofluorescence, has found HCG-like material on the sur-
* Supported by Grants from the Rockefeller Foundation (GA-HS-7406) and the National Institutes of Health Grant (HD-09004) from National Institutes of Health and World Health Organization. 1
2 Material and methods Studies on rabbits
Samples of HCG containing 12,000 IU/mg were provided by Dr. R.E. Canfield and Dr. O.P. Bahl. Aliquots of 10,100 and 200 pg of HCG in saline were impregnated into 1 mm X 1 cm implants provided by Dr. G. Gupta (Population Council, Rockefeller University, New York, N.Y). The HCG implants were inserted in the uterine horn of rabbits under aseptic conditions in the following manner. Rabbits were anesthesized with nembutal. A ventral incision was made through the vaginal wall. A 2-inch Tygon tube of 2 mm diameter was inserted into the uterus and the implant was then pushed into the uterine cavity via the indwelling Tygon tube. Care was taken to avoid any laceration of tissue around the cervix and inside the uterus. Sham operation was performed using implants without HCG. The blood was collected at 15and30minandat 1,2,and4handonthefollowing 3 days. On day 4, the animals were sacrificed; the implants were recovered from the uterus and the ovaries were removed. All serum samples were analyzed for HCG by the radioreceptorassay using plasma membrane of bovine corpora lutea of pregnancy and HCG labeled with 12’I by lactoperoxidase as described previously (Saxena et al., 1974). The blank, control and standards were made in serum containing basal levels of LH of equal protein concentration in the standard and unknown and to measure increase in luteotropic material over the basal levels. For example, 50 /J of rabbit serum, and 100/J of human serum were added to respective standards.
B.B.Saxena et al.: HCG transport through the reproductive tract
placed in the distal tubes and in the other in the more proximal portion close to the body of the uterus. In 2 final patients, HCG was deposited around the ovaries in the peritoneal cavity. Blood samples were obtained prior to and at 15 and 30 min and 1,2,4,8, 16, 24, and 48 h after the administration of HCG. The serum samples were analyzed for HCG by the radioreceptorassay.
Results and Discussion
The peripheral levels of HCG observed after implantation of 10, 100 and 200 pg in one uterine horn of the rabbit are shown in Figure 1. The HCG appeared in the blood as early as 15 min after implantation. From the three implants, HCG levels as high as 90, 165, and 625 ng/ml, respectively, were observed in the blood within 30 min to 2 h. On day 3, the blood levels were still elevated in the rabbits car-
b-ice ; NC/Ml
Studies in women
In one subject 5,000 IU of HCG was injected intramuscularly into the lateral buttock. An additional 7 women each received 5,000 IU of HCG in 2 ml of normal saline into various areas of the reproductive tract. In 3, HCG was instilled into the uterine cavity via a cervical cannula, after the tubes were tied at the uterotubal junction. The cannula remained in place for 1 h to maintain the HCG in contact with the endometrial surfaces. In 2, the HCG was instilled into the tubal lumen: 1 ml in the right and the second ml in the left. In 1 of these 2 patients, the HCG was
10
5
1
IMPLANT
TIME
24 ;
48
72
HOUR
Fig. 1. Detection of HCG in serum by radioreceptorassay following intrauterine implantation of HCG in rabbits. Sharnoperated rabbits showed no HCG in the serum.
3
B.B. Saxena et al.: HCG transport through the reproductive tract
rying implants of 100 and 200 pugHCG. The presence of up to 20-25 corpora lutea in the ovary (Fig. 2) demonstrated the HCG released by the implants crossed the uterine wall into the peripheral circulation and luteinized the ovaries. The sham-operated rabbits showed neither any HCG in the circulation nor stimulation of corpora lutea. When 5,000 IU HCG were injected intramuscularly, the highest plasma concentration occurred at 2 h (top channel; Fig. 3). Transport of HCG from the pelvic peritoneum in the ovarian region on the 6th and 8th days of the cycle was also detected at 8 h reaching a maximum between 16 and 24 h. After instillation of HCG on days 14 and 16, HCG was sustained up to a 48-h period in the blood. However, HCG was not detected in blood for up to 48 h when applied to the endosalpinx on days 10 and 26 of the cycle or to the endometrium on day 5. These results
Fig. 2. Superovulated ovary recovered 3 days following intrauterine implantation of 200 fig HCG in rabbit (left). The ovary from the sham-operated control rabbit is shown on the right.
I
I
ovary
1 ‘IIt
Day
6
Endometrium
Day
14
Endometrium
Day
15
,I Endometrium
10 I,;
Endosalpinx
0
-0
.l
Day Day
5 10 6 26
I
A
1 Hours
2 post
hCG
I
4
1
8
16
24
48
administration
Fig. 3. The detection of HCG in the serum following introduction of HCG into the endometrium, cavities as compared to intramuscular injection with 5,000 IU of HCG, in women.
endosalpinx,
and peritoneal
4
suggest that in the event a preimplantation HCG-like material is elaborated, it can cross the uterine cavities and reach the peripheral circulation. Methods to inactivate luteotropins by antibodies in early pregnancy are being considered as targets for contraception @Learn and Lunn, 1975; Stevens, 1973; Stevens and Crystle, 1973; Bahl, Pandyan and Moyle, 1975; Segal, 1976).
References Bahl, O.P., Pandyan, P.R., Moyle, W.R. and Kobayashi, Y. (1975): Chorionic gonadotropin in fertility and reproduction. In: Advances in Fertility Regulation through Basic Research. Editors: W.A. Sadler and S. Segal. Plenum Press, New York. Dickman, Z. and Dey, S.K. (1974): Evidence that A5-3p-hydroxysteroid dehydrogenase activity in rat blastocyst is autonomous.J. Endocr., 61, 513. Fujimoto, S., Euker, J.S., Riegel, G.D. and Dukelow, W.R. (1975): On a substance crossreacting with luteinizing hormone in the preimplantation blastocyst fluid of the rabbit. Proc. Japan Acad., 51, 123. Haour, F., Saxena, B.B. (1974): Detection of agonadotropin in rabbit blastocyst before implantation. Science, 185, 444. Haour, F., Charming, C. and Saxena, B.B. (1975): A stimula-
B.B. Saxena et al.: HCG transport through the reproductive tract tory effect of prelmplantation rabbit blastocyst fluid upon luteinization of monkey granulosa cell cultures. In: Abstracts, Endocrine Society 57th Annual Meeting, p. 107. Hearn, J.P. and Lunn, S.F. (1975): The endocrinology of the ovarian cycle and of pregnancy in the Maroset monkeys. J. Endocr., 65, 1. Kosasa, T.S., Levesque, L.A., Taymor, M.L. and Goldstein, D.P. (1974): Measurement of early chorionic activity with a radioimmunoassay specific for human chorionic gonadotropin following spontaneous and induced ovulation. Fertil. and Steril., 2.5, 2 11. Saxena, B.B., Hasan, S.H., Haour, F. and Schmidt-Gollwitzer, M. (1974): Radioreceptorassay of human chorionic gonadotropin: Detection of early pregnancy. Science 184, 793. Segal, S.J. (1976): Immunological methods to prevent pregnancy - an editorial comment. Contraception, 13, 125. Stevens, V.C. (1973): Immunization of female baboons with hapten coupled gonadotropins. Obstet. and Gynec., 42, 495. Stevens, V.C. and Crystle, C.D. (1973): Effects of immunization with hapten coupled hCG on the human menstrual cycle. Obstet. and Gynec., 42, 485. Sundaram, K., Connell, K.G., Passantino, T. (1975): Control of corpus Iuteum function in the pregnant rabbit: Absence of hCGlike material in the blastocyst. In: Abstracts,Endocrine Society 57th Annual Meeting, p. 107. Wiley, L.D. (1974): Presence of a gonadotropin on the surface of preimplanted mouse embryos. Nature (London), 252, 715.
