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Gonadotropin stimulation for in vitro fertilization and embryo transfer in insulin-dependent diabetics: follicular response, oocyte quality, embryo development, and follicular environment
Vol. 53, No.4, April 1990
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright e 1990 The American Fertility Society
Gonadotropin stimulation for in vitro fertilization and embryo transfer in insulin-dependent diabetics: follicular response, oocyte quality, embryo development, and follicular environment
Sergio Oehninger, M.D.* Glen E. Hofmann, M.D., Ph.D.t David Kreiner, M.D.:\:
Anibal A. Acosta, M.D. Suheil J. Muasher, M.D.
The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
In vitro fertilization (IVF) and embryo transfer (ET) is now an accepted modality for couples with a wide variety of reproductive disorders. In general, IVF patients are in good health, without major medical problems. To our knowledge there are no reports of women with insulin-dependent diabetes (IDD) undergoing gonadotropin stimulation for assisted reproduction. Diabetic animals have ovarian atrophy, poor follicular development, impaired steroidogenesis, and reduced progesterone (P) production, only partly restored by insulin administration.! Diamond et al. 2 reported that in diabetic mice the number of follicles recruited during gonadotropin stimulation was not reduced, but there was impairment in in vitro oocyte maturation and early pre-embryo development which was corrected by insulin administration. We report here two patients who underwent three IVF cycles, offering human models for the evaluation of ovarian response to gonadotropin stimulation, oocyte maturation and fertilization follicular hormonal milieu, and early embryo devel ~ opment in the IDD woman. Received August 28, 1989; revised and accepted December 21, 1989. * Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, 825 Fairfax Avenue, Norfolk, Vir· ginia 23507. t Present address: Department of Obstetrics and Gynecol· ogy, Mount Sinai Medical Center, New York, New York. Present address: Long Island IVF, Port Jefferson, New York.
*
Vol. 53, No.4, April 1990
CASE REPORTS
Two IDD patients presented for IVF; both had regular menstrual cycles. Patient 1 was 35 years old with a history of IDD since age 4, controlled with the daily administration of 40 U of NPH insulin (NPH iletin; Lilly Co, Indianapolis, IN). Her obstetric history included a second-trimester miscarriage (normal fetus) followed by two well-controlled pregnancies delivered at 36 weeks by cesarean section. She subsequently had a bilateral fimbriectomy for sterilization, with a failed reversal. Patient 2 was 36 years old with primary infertility of 2 years' duration, secondary to tubal adhesions. Diabetes was diagnosed 2 years before presentation, after a keto-acidotic episode. Insulin-dependent diabetes was controlled with 20 to 25 U of Lente insulin (Lilly Co.) in the morning and 15 U in the evening. Both patients had gonadotropin stimulation with a combination of follicle-stimulating hormone (FSH; Serono Laboratories, Randolph, MA) and human menopausal gonadotropin (Serono) starting on cycle day 3. Patient 2 also had a second cycle for pituitary suppression with leuprolide acetate (LA, Lupron; TAP Pharmaceuticals, North Chicago, IL) with the same combination protocol. 3 Follicles were aspirated transvaginally under ultrasound guidance until the follicles were completely collapsed. Oocytes were retrieved and classified according to the method of Veeck et al.,4 as imma~ Oehninger et al.
Communications· in-brief
741
Table 1
Hormonal and Oocyte Response to Gonadotropin Stimulation in Two IDD Patients· Basal
Cycle number
FSH
1 2 3
6.7 5.0 5.2
LH
mIU/mL
E2
PeakE2
Day of hCG
No. of ampules
Im/Preov oocytes b retrieved
Preov oocytes fertilized
10
13 18 23
2/14 1/5 1/12
10
pg/ml
5.0 7.2 10
40 40 11
2,500 950 1,134
10
12
• Patient 2 had a non-LA cycle (2) and a LA cycle (3).
b 1m, Immature oocyte with germinal vesicle (PI). Preov, Preovulatory oocyte without germinal vesicle (MI and MIl).
ture (prophase I [PI] with a germinal vesicle) or preovulatory (metaphase I [MI] or II [MIl]). In both cycles of Patient 2, follicular fluid (FF) volumes were measured; FF was taken to the laboratory on ice and centrifuged for 5 minutes at 2,000 X g to remove cellular debris. Follicular fluid was stored frozen at -20°C until assayed. Blood serum was separated and analyzed for FSH, luteinizing hormone (LH), and estradiol (E 2) on cycle day 3, and for E2 daily thereafter, using commercially available radioimmunoassay (RIA) kits (FSHQuant and LH-Quant; Leeco Diagnostics, Southfield, MI: E 2; Pantex, Santa Monica, CAl. The intra-assay and interassay coefficients of variation (CVs) for FSH, LH, and E2 were <7.5%. Examination of patient glycemic control was evaluated by measuring the fasting levels of glucose (glucose hexokinase method; Cobas ioanalyzer, Nutley, NJ), C-peptide (Incstar, Stillwater, MN), hemoglobin Ale (HbA lC ; BioRad, Richmond, CAl, and a 2-hour postprandial glucose. Intra-assay and interassay CVs were 1.2% and 2.6% for glucose, 7% and 11% for C-peptide, and 3.9% and 8.5% for HbA lC • Follicular fluid from follicles containing only MIl oocytes from both cycles of Patient 2 were analyzed for E 2, P (Pantex), androstenedione (A; Diagnostic Systems Laboratories, Webster, TX), human chorionic gonadotropin (hCG; Baxter Travenol Diagnostics, Cambridge, MA), prolactin (PRL; Diagnostic Products, Los Angeles, CAl, and epidermal growth factor (EGF; Biomedical Technologies, Stoughton, MA). Inadequate numbers of Table 2
E2 ng/mL
#C/mL
ng/mL
Non-LA LA,
432 ± 138 467 ± 279
2.6 ± 1.3 6.4 ± 3.2
90.6 ± 85.7 105.9 ± 84
P
RESULTS The results of gonadotropin stimulation for both patients (3 cycles) are shown in Table 1. All MI oocytes matured in vitro with extrusion of the first polar body. Normal fertilization rates were observed, and all embryos cleaved normally. Up to four embryos were transferred per cycle. Extra embryos were cryopreserved for transfer in a subsequent natural cycle. Post-thaw survival rate of cryopreserved embryos (3 of 4 in patient 1) was similar to our experience with non-IDD patients. Patient 2 still has six embryos cryopreserved. Neither patient became pregnant. Measurements of glycemic control indicated adequate metabolic control. Both patients had normal HbA lC , low-to-normal C-peptide, and normal fasting and 2-hour postprandial glucose levels. Analysis of FF from patient 2 is shown in Table 2. In none of the specimens could EGF or FSH be detected, even after repeated analysis. In our popu-
A
Values are expressed as means ± SD.
742
follicles containing PI or MI oocytes were available for FF analysis. All samples were run on the same day with intra-assay CVs of 7.6%, 5.1%, 7.4%, 5.2%, 5.9%, and 4.5% for the above hormones, respectively. The limit of detection for EGF was 0.25 ng/mL (50 pmoljL). Resulting data were compared with FF levels of the same FF constituents analyzed from a large group of non-IDD patients undergoing IVF during the same time frame (nonIDD controls). 5,6 All data are presented as the mean ±SD.
Hormonal Measurements from FF Obtained from Patient 2 in the Non-LA and LA cycles a
Cycle
a
3 11
Oehninger et al.
Communications-in-brief
hCG
b
PRL
FSH
EGF
mIU/mL
#C/ml
mIU/mL
ng/mL
62.6 ± 11.4 55.0 ± 27.7
33.5 ± 12.5 35.8 ± 15.2
ND b ND
ND ND
ND
=
not detectable.
Fertility and Sterility
lation of non-IDD patients, it is common for the level of FF FSH to be below the sensitivity level of the assay (5 mIU/mL). However, EGF was detected in all FF analyzed from non-IDD controls.5 Comparing levels of measured hormones between the LA (12 follicles) and non-LA (5 follicles) cycles of Patient 2 revealed only that P was higher in the LA cycle. Measured FF hormones (E 2, P, A, PRL, and hCG) from the LA cycle and non-LA cycles were similar to non-IDD controls (as compared using 95% confidence limits).6
DISCUSSION
Diamond et al.,2 in a preliminary report, noted no difference between IDD and non-IDD mice in the number of follicles recruited during gonadotropin stimulation. However, the IDD mice had a greater proportion of PI oocytes recovered than did non-IDD mice. The IDD mice also had fewer twocell embryos recovered after uterine flushing. Both of these situations were reversed by insulin. We report here the results of ovarian stimulation for IVF in two well-controlled IDD patients. Both had a high E2 response and a normal follicular recruitment (defined as the number of follicles recruited and oocytes retrieved). The ratio ofpreovulatory to immature oocytes was similar to our experience with our non-IDD population. 4 In addition, all embryos cleaved at rates comparable with the non-IDD population, and frozen-thawed embryos survived at rates similar to the non-IDD population. Thus, by the criteria of E2 response, oocyte number, maturity and fertilization rates, and early embryo development, IDD patients behaved similarly to non-IDD patients. Analysis of FF allowed a further assessment of the normalcy of the follicular milieu in IDD patients. We examined the FF hormones and factors in this study because of our large data base on nonIDD women in LA and non-LA cycles. 5,6 We attempted to measure insulinlike growth factor-I, but the heparinized Dulbecco's solution used for oocyte aspiration interfered with the RIA. Comparison of FF levels of the measured hormones from Patient 2 during LA and non-LA cycles revealed only that P was higher in the LA cycle, a result that differs from the non-IDD population. 6 In every follicle of the non-IDD group, EGF was identified, whereas no EGF was detectable in the FF from Patient 2. 5 Although data from a single patient is difficult to interpret, the absence ofEGF in FF (and its significance) deserves further exploVol. 53, No.4, April 1990
ration in light of recent evidence for a role of growth factors in ovarian function. We conclude that the well-controlled IDD patient has a normal response to gonadotropin stimulation with normal early in vitro embryo development and should be allowed to participate in IVF programs. SUMMARY
Two IDD patients were stimulated with gonadotropins for IVF and ET. Both patients had high E2 response and ~5 preovulatory oocytes retrieved, normal fertilization and cleavage rates, and transfer; neither conceived. Follicular fluid showed levels ofE2, P, A, hCG, and PRL similar to non-IDD. Epidermal growth factor could not be detected in FF. Insulin-dependent diabetes patients can undergo gonadotropin stimulation for IVF with customary responses. Their follicular milieu resembles that of non-IDD patients except for a lack ofEGF. Acknowledgment. We are grateful to Charlotte Schrader, Ph.D., for editorial assistance.
REFERENCES 1. Diamond MP, Wentz AC, Cherrington AD: Alterations in
2.
3.
4.
5.
6.
carbohydrate metabolism as they apply to reproductive endocrinology. Fertil Steril50:387, 1988 Diamond MP, Moley KH, Pellicer A, Vaughn WK, DeCherney AH: In vivo maturation and early embryo development in preimplantation embryos of non-diabetic and diabetic mice. (Abstr. 73) Presented at the Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17 to 20, 1988. Published in the program, p96 Droesch K, Muasher SJ, Brzyski RG, Jones GS, Simonetti S, Liu H -C, Rosenwaks Z: Value of suppression with a gonadotropin-releasing hormone agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil Steril51:292, 1989 Veeck LL, Wortham JWE, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW, Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39:594, 1983 Hofmann GE, Scott RT, Brzyski RG, Jones HW, Jr: Concentrations of immunoreactive epidermal growth factor (EGF) in follicular fluid obtained from in vitro fertilization: correlation with gonadal steroid concentrations and oocyte maturational status in cycles with and without the gonadotropin-releasing hormone agonist leuprolide acetate. Submitted Brzyski RG, Hofmann GE, Scott RT, Jones HW, Jr: Effects of leuprolide acetate on follicular fluid hormone composition at oocyte retrieval for in vitro fertilization. Submitted
Oebninger et at.
Communications-in-brief
743
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright e 1990 The American Fertility Society
Gonadotropin stimulation for in vitro fertilization and embryo transfer in insulin-dependent diabetics: follicular response, oocyte quality, embryo development, and follicular environment
Sergio Oehninger, M.D.* Glen E. Hofmann, M.D., Ph.D.t David Kreiner, M.D.:\:
Anibal A. Acosta, M.D. Suheil J. Muasher, M.D.
The Howard and Georgeanna Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
In vitro fertilization (IVF) and embryo transfer (ET) is now an accepted modality for couples with a wide variety of reproductive disorders. In general, IVF patients are in good health, without major medical problems. To our knowledge there are no reports of women with insulin-dependent diabetes (IDD) undergoing gonadotropin stimulation for assisted reproduction. Diabetic animals have ovarian atrophy, poor follicular development, impaired steroidogenesis, and reduced progesterone (P) production, only partly restored by insulin administration.! Diamond et al. 2 reported that in diabetic mice the number of follicles recruited during gonadotropin stimulation was not reduced, but there was impairment in in vitro oocyte maturation and early pre-embryo development which was corrected by insulin administration. We report here two patients who underwent three IVF cycles, offering human models for the evaluation of ovarian response to gonadotropin stimulation, oocyte maturation and fertilization follicular hormonal milieu, and early embryo devel ~ opment in the IDD woman. Received August 28, 1989; revised and accepted December 21, 1989. * Reprint requests: Sergio Oehninger, M.D., Jones Institute for Reproductive Medicine, 825 Fairfax Avenue, Norfolk, Vir· ginia 23507. t Present address: Department of Obstetrics and Gynecol· ogy, Mount Sinai Medical Center, New York, New York. Present address: Long Island IVF, Port Jefferson, New York.
*
Vol. 53, No.4, April 1990
CASE REPORTS
Two IDD patients presented for IVF; both had regular menstrual cycles. Patient 1 was 35 years old with a history of IDD since age 4, controlled with the daily administration of 40 U of NPH insulin (NPH iletin; Lilly Co, Indianapolis, IN). Her obstetric history included a second-trimester miscarriage (normal fetus) followed by two well-controlled pregnancies delivered at 36 weeks by cesarean section. She subsequently had a bilateral fimbriectomy for sterilization, with a failed reversal. Patient 2 was 36 years old with primary infertility of 2 years' duration, secondary to tubal adhesions. Diabetes was diagnosed 2 years before presentation, after a keto-acidotic episode. Insulin-dependent diabetes was controlled with 20 to 25 U of Lente insulin (Lilly Co.) in the morning and 15 U in the evening. Both patients had gonadotropin stimulation with a combination of follicle-stimulating hormone (FSH; Serono Laboratories, Randolph, MA) and human menopausal gonadotropin (Serono) starting on cycle day 3. Patient 2 also had a second cycle for pituitary suppression with leuprolide acetate (LA, Lupron; TAP Pharmaceuticals, North Chicago, IL) with the same combination protocol. 3 Follicles were aspirated transvaginally under ultrasound guidance until the follicles were completely collapsed. Oocytes were retrieved and classified according to the method of Veeck et al.,4 as imma~ Oehninger et al.
Communications· in-brief
741
Table 1
Hormonal and Oocyte Response to Gonadotropin Stimulation in Two IDD Patients· Basal
Cycle number
FSH
1 2 3
6.7 5.0 5.2
LH
mIU/mL
E2
PeakE2
Day of hCG
No. of ampules
Im/Preov oocytes b retrieved
Preov oocytes fertilized
10
13 18 23
2/14 1/5 1/12
10
pg/ml
5.0 7.2 10
40 40 11
2,500 950 1,134
10
12
• Patient 2 had a non-LA cycle (2) and a LA cycle (3).
b 1m, Immature oocyte with germinal vesicle (PI). Preov, Preovulatory oocyte without germinal vesicle (MI and MIl).
ture (prophase I [PI] with a germinal vesicle) or preovulatory (metaphase I [MI] or II [MIl]). In both cycles of Patient 2, follicular fluid (FF) volumes were measured; FF was taken to the laboratory on ice and centrifuged for 5 minutes at 2,000 X g to remove cellular debris. Follicular fluid was stored frozen at -20°C until assayed. Blood serum was separated and analyzed for FSH, luteinizing hormone (LH), and estradiol (E 2) on cycle day 3, and for E2 daily thereafter, using commercially available radioimmunoassay (RIA) kits (FSHQuant and LH-Quant; Leeco Diagnostics, Southfield, MI: E 2; Pantex, Santa Monica, CAl. The intra-assay and interassay coefficients of variation (CVs) for FSH, LH, and E2 were <7.5%. Examination of patient glycemic control was evaluated by measuring the fasting levels of glucose (glucose hexokinase method; Cobas ioanalyzer, Nutley, NJ), C-peptide (Incstar, Stillwater, MN), hemoglobin Ale (HbA lC ; BioRad, Richmond, CAl, and a 2-hour postprandial glucose. Intra-assay and interassay CVs were 1.2% and 2.6% for glucose, 7% and 11% for C-peptide, and 3.9% and 8.5% for HbA lC • Follicular fluid from follicles containing only MIl oocytes from both cycles of Patient 2 were analyzed for E 2, P (Pantex), androstenedione (A; Diagnostic Systems Laboratories, Webster, TX), human chorionic gonadotropin (hCG; Baxter Travenol Diagnostics, Cambridge, MA), prolactin (PRL; Diagnostic Products, Los Angeles, CAl, and epidermal growth factor (EGF; Biomedical Technologies, Stoughton, MA). Inadequate numbers of Table 2
E2 ng/mL
#C/mL
ng/mL
Non-LA LA,
432 ± 138 467 ± 279
2.6 ± 1.3 6.4 ± 3.2
90.6 ± 85.7 105.9 ± 84
P
RESULTS The results of gonadotropin stimulation for both patients (3 cycles) are shown in Table 1. All MI oocytes matured in vitro with extrusion of the first polar body. Normal fertilization rates were observed, and all embryos cleaved normally. Up to four embryos were transferred per cycle. Extra embryos were cryopreserved for transfer in a subsequent natural cycle. Post-thaw survival rate of cryopreserved embryos (3 of 4 in patient 1) was similar to our experience with non-IDD patients. Patient 2 still has six embryos cryopreserved. Neither patient became pregnant. Measurements of glycemic control indicated adequate metabolic control. Both patients had normal HbA lC , low-to-normal C-peptide, and normal fasting and 2-hour postprandial glucose levels. Analysis of FF from patient 2 is shown in Table 2. In none of the specimens could EGF or FSH be detected, even after repeated analysis. In our popu-
A
Values are expressed as means ± SD.
742
follicles containing PI or MI oocytes were available for FF analysis. All samples were run on the same day with intra-assay CVs of 7.6%, 5.1%, 7.4%, 5.2%, 5.9%, and 4.5% for the above hormones, respectively. The limit of detection for EGF was 0.25 ng/mL (50 pmoljL). Resulting data were compared with FF levels of the same FF constituents analyzed from a large group of non-IDD patients undergoing IVF during the same time frame (nonIDD controls). 5,6 All data are presented as the mean ±SD.
Hormonal Measurements from FF Obtained from Patient 2 in the Non-LA and LA cycles a
Cycle
a
3 11
Oehninger et al.
Communications-in-brief
hCG
b
PRL
FSH
EGF
mIU/mL
#C/ml
mIU/mL
ng/mL
62.6 ± 11.4 55.0 ± 27.7
33.5 ± 12.5 35.8 ± 15.2
ND b ND
ND ND
ND
=
not detectable.
Fertility and Sterility
lation of non-IDD patients, it is common for the level of FF FSH to be below the sensitivity level of the assay (5 mIU/mL). However, EGF was detected in all FF analyzed from non-IDD controls.5 Comparing levels of measured hormones between the LA (12 follicles) and non-LA (5 follicles) cycles of Patient 2 revealed only that P was higher in the LA cycle. Measured FF hormones (E 2, P, A, PRL, and hCG) from the LA cycle and non-LA cycles were similar to non-IDD controls (as compared using 95% confidence limits).6
DISCUSSION
Diamond et al.,2 in a preliminary report, noted no difference between IDD and non-IDD mice in the number of follicles recruited during gonadotropin stimulation. However, the IDD mice had a greater proportion of PI oocytes recovered than did non-IDD mice. The IDD mice also had fewer twocell embryos recovered after uterine flushing. Both of these situations were reversed by insulin. We report here the results of ovarian stimulation for IVF in two well-controlled IDD patients. Both had a high E2 response and a normal follicular recruitment (defined as the number of follicles recruited and oocytes retrieved). The ratio ofpreovulatory to immature oocytes was similar to our experience with our non-IDD population. 4 In addition, all embryos cleaved at rates comparable with the non-IDD population, and frozen-thawed embryos survived at rates similar to the non-IDD population. Thus, by the criteria of E2 response, oocyte number, maturity and fertilization rates, and early embryo development, IDD patients behaved similarly to non-IDD patients. Analysis of FF allowed a further assessment of the normalcy of the follicular milieu in IDD patients. We examined the FF hormones and factors in this study because of our large data base on nonIDD women in LA and non-LA cycles. 5,6 We attempted to measure insulinlike growth factor-I, but the heparinized Dulbecco's solution used for oocyte aspiration interfered with the RIA. Comparison of FF levels of the measured hormones from Patient 2 during LA and non-LA cycles revealed only that P was higher in the LA cycle, a result that differs from the non-IDD population. 6 In every follicle of the non-IDD group, EGF was identified, whereas no EGF was detectable in the FF from Patient 2. 5 Although data from a single patient is difficult to interpret, the absence ofEGF in FF (and its significance) deserves further exploVol. 53, No.4, April 1990
ration in light of recent evidence for a role of growth factors in ovarian function. We conclude that the well-controlled IDD patient has a normal response to gonadotropin stimulation with normal early in vitro embryo development and should be allowed to participate in IVF programs. SUMMARY
Two IDD patients were stimulated with gonadotropins for IVF and ET. Both patients had high E2 response and ~5 preovulatory oocytes retrieved, normal fertilization and cleavage rates, and transfer; neither conceived. Follicular fluid showed levels ofE2, P, A, hCG, and PRL similar to non-IDD. Epidermal growth factor could not be detected in FF. Insulin-dependent diabetes patients can undergo gonadotropin stimulation for IVF with customary responses. Their follicular milieu resembles that of non-IDD patients except for a lack ofEGF. Acknowledgment. We are grateful to Charlotte Schrader, Ph.D., for editorial assistance.
REFERENCES 1. Diamond MP, Wentz AC, Cherrington AD: Alterations in
2.
3.
4.
5.
6.
carbohydrate metabolism as they apply to reproductive endocrinology. Fertil Steril50:387, 1988 Diamond MP, Moley KH, Pellicer A, Vaughn WK, DeCherney AH: In vivo maturation and early embryo development in preimplantation embryos of non-diabetic and diabetic mice. (Abstr. 73) Presented at the Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17 to 20, 1988. Published in the program, p96 Droesch K, Muasher SJ, Brzyski RG, Jones GS, Simonetti S, Liu H -C, Rosenwaks Z: Value of suppression with a gonadotropin-releasing hormone agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil Steril51:292, 1989 Veeck LL, Wortham JWE, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW, Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39:594, 1983 Hofmann GE, Scott RT, Brzyski RG, Jones HW, Jr: Concentrations of immunoreactive epidermal growth factor (EGF) in follicular fluid obtained from in vitro fertilization: correlation with gonadal steroid concentrations and oocyte maturational status in cycles with and without the gonadotropin-releasing hormone agonist leuprolide acetate. Submitted Brzyski RG, Hofmann GE, Scott RT, Jones HW, Jr: Effects of leuprolide acetate on follicular fluid hormone composition at oocyte retrieval for in vitro fertilization. Submitted
Oebninger et at.
Communications-in-brief
743