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Artificially induced menstrual cycle with natural estradiol and progesterone*
FERTILITY AND STERILITY
Vol. 51, No.6, June 1989 Printed in U.S.A.
Copyright c 1989 The American Fertility Society
Artificially induced menstrual cycle with natural estradiol and progesterone* Terry T. Hung, M.D., Ph.D.t:l: Delia Ribas, M.D. t Akira Tsuiki, M.D.t
Janet Preyer, B.Sc.t§ Robert Slackman, M.D. t Oscar W. Davidson, M.D. t
University of Miami School of Medicine, Reproductive Sciences and Endocrinology Laboratories (REPSCEND), Miami, Florida
In order to study the feasibility and efficacy of using natural 17,8-estradiol (E2) and progesterone (P) to induce endometrial changes, a group of patients with history of premature ovarian failure and bilateral oophorectomy, also interested in the embryo transfer program with donor ovum, were given transdermal E 2 (Estraderm, Ciba Pharmaceutical Co., Summit, NJ), and vaginal progesterone suppositories. Serial serum E 2 , P, folliclestimulating hormone, and luteinizing hormone were determined by radioimmunoassays. The dosages of E 2 and P were adjusted according to the levels of E 2 and P so that their changes could follow the same pattern as that of a normal spontaneous menstrual cycle. Serial ultrasonic evaluation of the endometrium and endometrial biopsy during the late luteal phase also was performed. Preliminary data indicated that transdermal E 2 patches and vaginal P suppositories, while being as effective in inducing endometrial development for the embryo transfer procedure with donor ovum as synthetic steroids, can also provide a more physiologic approach that may conveniently and safely be extended into the second trimester of pregnancy. Fertil Steril51:968, 1989
The incidence of women with premature ovarian failure, natural or surgical, based on current data, 1 is 86 out of 100,000 women yearly. In a majority of these women, the ovaries were atrophic and devoid of follicles; therefore, permanent sterility was unavoidable. Recently, however, with the establishment of in vitro fertilization (IVF) and human embryo transfer (ET) procedures, pregnancies have been accomplished by the induction of an artificial cycle on the recipient mother and the transfer of donated embryos or donated oocytes, later fertil-
Received August 11, 1988; revised and accepted February 15, 1989. "Presented at the Forty-Third Annual Meeting of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada. t Department of Obstetrics and Gynecology. :J: Reprint requests: Terry T. Hung, M.D., Ph.D., Associate Professor, University of Miami School of Medicine, Department of Obstetrics and Gynecology, P.O. Box 016960, Miami, Florida 33101. § REPSCEND Laboratory.
968
Hung et al. ArtificiaUy induced menstrual cycle
ized by sperm from the recipient's husband, into the recipient's uterus. The technique of ovulation induction, oocyte retrieval, IVF, embryo culture, and ET remains the same as with the standard IVF procedure performed on a patient with intact ovaries. One of the most important aspects of this procedure is to induce the recipient mother with exogenous estrogen and progestins (P) so that her endometrium can reach a stage that is not only susceptible to implantation but also is synchronized at the time of the ET with embryos obtained from the donor. Different regimens to induce artifically an endometrial cycle in the recipient have been tried successfully. 2- 5 However, most of the estrogen and progestins used in these regimens have been either synthetic or conjugated. Such administered steroids could have undergone metabolism in various organs, which might lead to some adverse effects on the recipient patient.6 Therefore, the purpose of this study was to circumvent these problems by investigating the feasibility and efficacy of using only natural 17,8-estradiol (E2 ) percutaneously and n.atural P intravaginally to induce endoFertility and Sterility
metrial changes in a group of patients with a history of premature ovarian failure who also were interested in the ET program with the ovum donor.
0.8
VARIABLE REGIMEN (n
= 3)
0.5
o.• 0.3 0.2
MATERIALS AND METHODS
0.1 FIXED REGIMEN (n
Patient Selection
=•l
o.•
Couples in which the woman had premature ovarian failure and was interested in the IVF procedure with donor ovum were recruited for this study (one with spontaneous premature ovarian failure, three after bilateral oophorectomy). Their ages varied between 21 and 36 years, and they all had four weekly serum follicle-stimulating hormone (FSH determination) with levels > 80 miUI ml and serum E 2 level < 40 pg/ml. Physical examination showed all of the women to be in good health and suffering no major contraindication for steroid-replacement therapy. Semen analysis, hysterosalpingogram, and endometrial biopsy had been performed to evaluate their eligibility for the IVF program. They were given a thorough explanation of all aspects of the treatment and signed informed consent forms before the initiation of the study. Medication
Estradiol was given percutaneously through the use of a transdermal delivery system, which was manufactured to deliver 50 11-g to 100 11-g per patch (Estraderm, Ciba Pharmaceutical Co., Summit, NJ). Progesterone suppositories were prepared from a mixture of crystalline P and polyethyleneglycol by the local pharmacy. The dosage of P per suppository ranged between 25 and 100 mg. Protocol
All other forms of estrogen replacement therapy were discontinued in the patients before the initiation of the treatment cycle. A baseline luteinizing hormone (LH), FSH, E 2 , and P also were performed. Two regimens were given to the patients, as described in Figure 1. In the variable regimen, the patient was started with 100 11-g of transdermal E 2 from day 1 on. The dosage of E 2 was adjusted according to the levels of FSH, LH, and E 2 determined in her serum 24 hours after the application of the medication by radioimmunoassays (RIA) in order to achieve E 2 levels comparable to those measured in spontaneous ovulatory cycles. The dosage of E 2 can reach 600 11-g near the middle of the cycle in order to create an E 2 surge. Then the dose was Vol. 51, No.6, June 1989
0.3
ESTAADEAM
0.2 0.1
CYCLE DAY
Figure 1 Variation of estrogen and progesterone dosage throughout the artificial cycles with variable and fixed regimens.
reduced to <300 11-g during the secretory phase. Progesterone vaginal suppositories were given between 25 and 100 mg per day after the onset of the LH surge in order to achieve P levels comparable to those measured during the luteal phase. The dosage of E 2 and P was adjusted according to the level of serum E 2 and P. In the fixed regimen, patients received a fixed dose ofE 2 (Estraderm), 200 11-g twice weekly for the first 2 weeks and then 400 11-g twice weekly for the following 2 weeks. Progesterone suppositories were given at a fixed dose of 100 mg from day 16 to day 28 of the cycle. Serum was collected by venipuncture every third day during the early menstrual cycle and daily during the midcycle and during the secretory phase. Serum LH, FSH, E 2 , and P were determined by RIA. Serial ultrasound evaluation of the endometrial lining was performed with an ADR vaginal probe attached to an ADR 4000 S/L realtime ultrasound machine (ADR, Bellingham, W A). An endometrial biopsy was performed during the late secretory phase by using a Curelle curette (Unimar, Wilton, CT) while the patient was anesthetized with a paracervical block using lidocaine 1%. Endometrial dating was performed according to the criteria of Noyes et al. 7 RESULTS
Figure 2 shows the change in serum E 2 , P, FSH, and LH throughout the treatment cycle with the variable regimen among the three patients. The bars indicate the standard error of the mean. The serum E 2 surge was accomplished around day 14 of the treatment cycle with a level ranging between 600 and 900 pg/ml. A second surge ofE 2 during the Hung et al.
Artificially induced menstrual cycle
969
VARIABLE REGIMEN (n
:i1000 :IE
=3)
~ :IE
OESTRADIOL
"..
0 PROGESTERONE
"
!
~ 800
10 ~
......,_
-'
0
~
.. ,_a:
a:
&00
"0
w 400
:IE ::>
5
a: ~ 200
:: :IE ::>
... a:
CYCLE DAY
Figure 2 Serum concentrations of E 2 , P, LH, and FSH throughout the artificial menstrual cycles induced by a variable regimen.
midsecretory phase between 300 and 500 pg/ml also had been achieved. Serum P increased steadfastly after the E 2 surge, and a peak above 10 ng/ ml was observed during the middle of the secretory phase. Surprisingly, the LH and FSH surge was observed in all three patients and occurred 24 hours after the achievement of the E 2 surge. The baseline of LH and FSH levels during the secretory phase was significantly lower than during the proliferative phase of the cycle, probably because of the negative feedback effects of P in addition to those of E 2 • Endometrial evaluation by ultrasound throughout the whole cycle showed a gradual increase in the thickness from a 5 mm up to an average of 13 mm in thickness during the midsecretory phase. The midline echo was observed usually during the late proliferative phase or early secretory phase, and was much more prominent around the time ofE2 and LH surge. However, in one patient, there was difficulty in obtaining a clearly defined endometrium in contrast with her satisfactory serum E 2 and P levels. Figure 3 shows the change of serum E 2 , P, LH, and FSH among the four patients undergoing a fixed regimen. There was no E 2 surge, as well as no LH and FSH surge observed at the ~idcycle. However, endometrial evaluation by ultrasound showed satisfactory development comparable with those patients in the variable regimen .. 970
Endometrial biopsy was performed on all patients in both regimens and was done between day 23 and day 28 of the cycle, which indicated late secretory phase endometrium. All dating fell within 2 days from the day ofLH surge with the variable regimen and within 2 days from the day of initiation of the treatment with the fixed regimen .
Bung et al. Artificially induced menstrual cycle
DISCUSSION
This is the first study indicating the feasibility of using natural E 2 and P transdermally and transvaginally to induce adequate response in the endometrial tissues susceptible for implantation if ET is to be performed. However, these conclusions are based mainly on ultrasound and histologic evaluation ofthe endometrium. We are still waiting for a successful pregnancy in order to verify the ultimate goal of this approach. Meanwhile, this approach is very promising because of certain advantages: (1) it would provide a simple, noninvasive, less complicated means of administration of exogenous steroids that the patients can administer to themselves with ease. The patients undergoing this type of treatment have responded positively regarding its simplicity, convenience, and the low incidence of side effects or complications; and (2) it provides a means to administer a natural, nonsynthetic or conjugated estrogen directly into the circulation without going first through the enterohepatic circulation, thereby avoiding some of the potentially adverse effects of orally administered estrogen and progestin on the serum lipoproteins profile.8 Finally, there was no difference in the endometrial response from both ultrasonic and histologic asFIXED REGIMEN
..• -'
eoo
!. -'
ac
400
1-
200
0
10
. ! z
.. .•..
0
0: 1-
a:
.•
•. -'
(n • 4)
OESTRADIOL c PROGESTERONE
.. 0
0:
-'
~
::>
!
40
i
20
•z
0
0: 1-
0 0
c
. z
0
~ _________ .....,__________
10
12
18
20
24
. 28
CYCLE DAY
Figure 3 Serum concentrations of E 2 , P, LH, and FSH throughout the artificial menstrual cycles induced by a fixed regimen. Fertility and Sterility
pects between patients using fixed and variable regimens. Because of its simplicity, the fixed regimen may be a preferable choice of treatment in the future for the patient undergoing this type of procedure; however, this preliminary study needs to be investigated further, and successful embryo implantation must be demonstrated before its efficacy can be compared with other regimens currently used to induce an artificial menstrual cycle.
3.
4.
5.
6.
REFERENCES 7. 1. Coulam CB, Adamson SC, Annergero JF: Incidence of pre-
mature ovarian failure. Obstet Gynecol 67:604, 1986 2. Nanist D, Laufer N, Kopolovic J, Rabinowitz R, Birkenfeld A, Lewin A, Granat M, Margalioth E, Schenker J: Artificially induced endometrial cycles and establishment of
Vol. 51, No.6, June 1989
8.
pregnancies in the absence of ovaries. N Engl J Med 324: 806, 1986 Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P: The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 307:174, 1984 Bustillo M, Buster JE, Cohen SW: Nonsurgical ovum transfer as a treatment in infertile women: preliminary experience. JAMA 251:1,171, 1984 Hodgen GD: Surrogate embryo transfer combined with estrogen-progesterone therapy in monkeys: implantation, gestation and delivery without ovaries. JAMA 250:2,176, 1983 Ryan KJ: Placental synthesis of steroid hormones. In Maternal-Fetal Endocrinology, Edited by D Tulchinsky, KJ Ryan. Philadelphia, WB Saunders, 1980, p 3 Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, 1950 Jensen J, Riis BJ, Stron V: Long-term effects of percutaneous estrogens and oral progesterone on serum lipoproteins in postmenopausal women. Am J Obstet Gynecol 156:66, 1987
Hung et al.
Artificially induced menstrual cycle
971
Vol. 51, No.6, June 1989 Printed in U.S.A.
Copyright c 1989 The American Fertility Society
Artificially induced menstrual cycle with natural estradiol and progesterone* Terry T. Hung, M.D., Ph.D.t:l: Delia Ribas, M.D. t Akira Tsuiki, M.D.t
Janet Preyer, B.Sc.t§ Robert Slackman, M.D. t Oscar W. Davidson, M.D. t
University of Miami School of Medicine, Reproductive Sciences and Endocrinology Laboratories (REPSCEND), Miami, Florida
In order to study the feasibility and efficacy of using natural 17,8-estradiol (E2) and progesterone (P) to induce endometrial changes, a group of patients with history of premature ovarian failure and bilateral oophorectomy, also interested in the embryo transfer program with donor ovum, were given transdermal E 2 (Estraderm, Ciba Pharmaceutical Co., Summit, NJ), and vaginal progesterone suppositories. Serial serum E 2 , P, folliclestimulating hormone, and luteinizing hormone were determined by radioimmunoassays. The dosages of E 2 and P were adjusted according to the levels of E 2 and P so that their changes could follow the same pattern as that of a normal spontaneous menstrual cycle. Serial ultrasonic evaluation of the endometrium and endometrial biopsy during the late luteal phase also was performed. Preliminary data indicated that transdermal E 2 patches and vaginal P suppositories, while being as effective in inducing endometrial development for the embryo transfer procedure with donor ovum as synthetic steroids, can also provide a more physiologic approach that may conveniently and safely be extended into the second trimester of pregnancy. Fertil Steril51:968, 1989
The incidence of women with premature ovarian failure, natural or surgical, based on current data, 1 is 86 out of 100,000 women yearly. In a majority of these women, the ovaries were atrophic and devoid of follicles; therefore, permanent sterility was unavoidable. Recently, however, with the establishment of in vitro fertilization (IVF) and human embryo transfer (ET) procedures, pregnancies have been accomplished by the induction of an artificial cycle on the recipient mother and the transfer of donated embryos or donated oocytes, later fertil-
Received August 11, 1988; revised and accepted February 15, 1989. "Presented at the Forty-Third Annual Meeting of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada. t Department of Obstetrics and Gynecology. :J: Reprint requests: Terry T. Hung, M.D., Ph.D., Associate Professor, University of Miami School of Medicine, Department of Obstetrics and Gynecology, P.O. Box 016960, Miami, Florida 33101. § REPSCEND Laboratory.
968
Hung et al. ArtificiaUy induced menstrual cycle
ized by sperm from the recipient's husband, into the recipient's uterus. The technique of ovulation induction, oocyte retrieval, IVF, embryo culture, and ET remains the same as with the standard IVF procedure performed on a patient with intact ovaries. One of the most important aspects of this procedure is to induce the recipient mother with exogenous estrogen and progestins (P) so that her endometrium can reach a stage that is not only susceptible to implantation but also is synchronized at the time of the ET with embryos obtained from the donor. Different regimens to induce artifically an endometrial cycle in the recipient have been tried successfully. 2- 5 However, most of the estrogen and progestins used in these regimens have been either synthetic or conjugated. Such administered steroids could have undergone metabolism in various organs, which might lead to some adverse effects on the recipient patient.6 Therefore, the purpose of this study was to circumvent these problems by investigating the feasibility and efficacy of using only natural 17,8-estradiol (E2 ) percutaneously and n.atural P intravaginally to induce endoFertility and Sterility
metrial changes in a group of patients with a history of premature ovarian failure who also were interested in the ET program with the ovum donor.
0.8
VARIABLE REGIMEN (n
= 3)
0.5
o.• 0.3 0.2
MATERIALS AND METHODS
0.1 FIXED REGIMEN (n
Patient Selection
=•l
o.•
Couples in which the woman had premature ovarian failure and was interested in the IVF procedure with donor ovum were recruited for this study (one with spontaneous premature ovarian failure, three after bilateral oophorectomy). Their ages varied between 21 and 36 years, and they all had four weekly serum follicle-stimulating hormone (FSH determination) with levels > 80 miUI ml and serum E 2 level < 40 pg/ml. Physical examination showed all of the women to be in good health and suffering no major contraindication for steroid-replacement therapy. Semen analysis, hysterosalpingogram, and endometrial biopsy had been performed to evaluate their eligibility for the IVF program. They were given a thorough explanation of all aspects of the treatment and signed informed consent forms before the initiation of the study. Medication
Estradiol was given percutaneously through the use of a transdermal delivery system, which was manufactured to deliver 50 11-g to 100 11-g per patch (Estraderm, Ciba Pharmaceutical Co., Summit, NJ). Progesterone suppositories were prepared from a mixture of crystalline P and polyethyleneglycol by the local pharmacy. The dosage of P per suppository ranged between 25 and 100 mg. Protocol
All other forms of estrogen replacement therapy were discontinued in the patients before the initiation of the treatment cycle. A baseline luteinizing hormone (LH), FSH, E 2 , and P also were performed. Two regimens were given to the patients, as described in Figure 1. In the variable regimen, the patient was started with 100 11-g of transdermal E 2 from day 1 on. The dosage of E 2 was adjusted according to the levels of FSH, LH, and E 2 determined in her serum 24 hours after the application of the medication by radioimmunoassays (RIA) in order to achieve E 2 levels comparable to those measured in spontaneous ovulatory cycles. The dosage of E 2 can reach 600 11-g near the middle of the cycle in order to create an E 2 surge. Then the dose was Vol. 51, No.6, June 1989
0.3
ESTAADEAM
0.2 0.1
CYCLE DAY
Figure 1 Variation of estrogen and progesterone dosage throughout the artificial cycles with variable and fixed regimens.
reduced to <300 11-g during the secretory phase. Progesterone vaginal suppositories were given between 25 and 100 mg per day after the onset of the LH surge in order to achieve P levels comparable to those measured during the luteal phase. The dosage of E 2 and P was adjusted according to the level of serum E 2 and P. In the fixed regimen, patients received a fixed dose ofE 2 (Estraderm), 200 11-g twice weekly for the first 2 weeks and then 400 11-g twice weekly for the following 2 weeks. Progesterone suppositories were given at a fixed dose of 100 mg from day 16 to day 28 of the cycle. Serum was collected by venipuncture every third day during the early menstrual cycle and daily during the midcycle and during the secretory phase. Serum LH, FSH, E 2 , and P were determined by RIA. Serial ultrasound evaluation of the endometrial lining was performed with an ADR vaginal probe attached to an ADR 4000 S/L realtime ultrasound machine (ADR, Bellingham, W A). An endometrial biopsy was performed during the late secretory phase by using a Curelle curette (Unimar, Wilton, CT) while the patient was anesthetized with a paracervical block using lidocaine 1%. Endometrial dating was performed according to the criteria of Noyes et al. 7 RESULTS
Figure 2 shows the change in serum E 2 , P, FSH, and LH throughout the treatment cycle with the variable regimen among the three patients. The bars indicate the standard error of the mean. The serum E 2 surge was accomplished around day 14 of the treatment cycle with a level ranging between 600 and 900 pg/ml. A second surge ofE 2 during the Hung et al.
Artificially induced menstrual cycle
969
VARIABLE REGIMEN (n
:i1000 :IE
=3)
~ :IE
OESTRADIOL
"..
0 PROGESTERONE
"
!
~ 800
10 ~
......,_
-'
0
~
.. ,_a:
a:
&00
"0
w 400
:IE ::>
5
a: ~ 200
:: :IE ::>
... a:
CYCLE DAY
Figure 2 Serum concentrations of E 2 , P, LH, and FSH throughout the artificial menstrual cycles induced by a variable regimen.
midsecretory phase between 300 and 500 pg/ml also had been achieved. Serum P increased steadfastly after the E 2 surge, and a peak above 10 ng/ ml was observed during the middle of the secretory phase. Surprisingly, the LH and FSH surge was observed in all three patients and occurred 24 hours after the achievement of the E 2 surge. The baseline of LH and FSH levels during the secretory phase was significantly lower than during the proliferative phase of the cycle, probably because of the negative feedback effects of P in addition to those of E 2 • Endometrial evaluation by ultrasound throughout the whole cycle showed a gradual increase in the thickness from a 5 mm up to an average of 13 mm in thickness during the midsecretory phase. The midline echo was observed usually during the late proliferative phase or early secretory phase, and was much more prominent around the time ofE2 and LH surge. However, in one patient, there was difficulty in obtaining a clearly defined endometrium in contrast with her satisfactory serum E 2 and P levels. Figure 3 shows the change of serum E 2 , P, LH, and FSH among the four patients undergoing a fixed regimen. There was no E 2 surge, as well as no LH and FSH surge observed at the ~idcycle. However, endometrial evaluation by ultrasound showed satisfactory development comparable with those patients in the variable regimen .. 970
Endometrial biopsy was performed on all patients in both regimens and was done between day 23 and day 28 of the cycle, which indicated late secretory phase endometrium. All dating fell within 2 days from the day ofLH surge with the variable regimen and within 2 days from the day of initiation of the treatment with the fixed regimen .
Bung et al. Artificially induced menstrual cycle
DISCUSSION
This is the first study indicating the feasibility of using natural E 2 and P transdermally and transvaginally to induce adequate response in the endometrial tissues susceptible for implantation if ET is to be performed. However, these conclusions are based mainly on ultrasound and histologic evaluation ofthe endometrium. We are still waiting for a successful pregnancy in order to verify the ultimate goal of this approach. Meanwhile, this approach is very promising because of certain advantages: (1) it would provide a simple, noninvasive, less complicated means of administration of exogenous steroids that the patients can administer to themselves with ease. The patients undergoing this type of treatment have responded positively regarding its simplicity, convenience, and the low incidence of side effects or complications; and (2) it provides a means to administer a natural, nonsynthetic or conjugated estrogen directly into the circulation without going first through the enterohepatic circulation, thereby avoiding some of the potentially adverse effects of orally administered estrogen and progestin on the serum lipoproteins profile.8 Finally, there was no difference in the endometrial response from both ultrasonic and histologic asFIXED REGIMEN
..• -'
eoo
!. -'
ac
400
1-
200
0
10
. ! z
.. .•..
0
0: 1-
a:
.•
•. -'
(n • 4)
OESTRADIOL c PROGESTERONE
.. 0
0:
-'
~
::>
!
40
i
20
•z
0
0: 1-
0 0
c
. z
0
~ _________ .....,__________
10
12
18
20
24
. 28
CYCLE DAY
Figure 3 Serum concentrations of E 2 , P, LH, and FSH throughout the artificial menstrual cycles induced by a fixed regimen. Fertility and Sterility
pects between patients using fixed and variable regimens. Because of its simplicity, the fixed regimen may be a preferable choice of treatment in the future for the patient undergoing this type of procedure; however, this preliminary study needs to be investigated further, and successful embryo implantation must be demonstrated before its efficacy can be compared with other regimens currently used to induce an artificial menstrual cycle.
3.
4.
5.
6.
REFERENCES 7. 1. Coulam CB, Adamson SC, Annergero JF: Incidence of pre-
mature ovarian failure. Obstet Gynecol 67:604, 1986 2. Nanist D, Laufer N, Kopolovic J, Rabinowitz R, Birkenfeld A, Lewin A, Granat M, Margalioth E, Schenker J: Artificially induced endometrial cycles and establishment of
Vol. 51, No.6, June 1989
8.
pregnancies in the absence of ovaries. N Engl J Med 324: 806, 1986 Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P: The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 307:174, 1984 Bustillo M, Buster JE, Cohen SW: Nonsurgical ovum transfer as a treatment in infertile women: preliminary experience. JAMA 251:1,171, 1984 Hodgen GD: Surrogate embryo transfer combined with estrogen-progesterone therapy in monkeys: implantation, gestation and delivery without ovaries. JAMA 250:2,176, 1983 Ryan KJ: Placental synthesis of steroid hormones. In Maternal-Fetal Endocrinology, Edited by D Tulchinsky, KJ Ryan. Philadelphia, WB Saunders, 1980, p 3 Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Fertil Steril1:3, 1950 Jensen J, Riis BJ, Stron V: Long-term effects of percutaneous estrogens and oral progesterone on serum lipoproteins in postmenopausal women. Am J Obstet Gynecol 156:66, 1987
Hung et al.
Artificially induced menstrual cycle
971