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Cytogenetic evaluation of human oocytes that failed to complete meiotic maturation in vitro
FERTILITY AND STERILITY威 VOL. 77, NO. 4, APRIL 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Cytogenetic evaluation of human oocytes that failed to complete meiotic maturation in vitro Michael S. Neal, M.Sc.,a Lisa Cowan, R.T.,a Jackie Pierre Louis, B.Sc.,a Ed Hughes, M.D.,a W. Allan King, Ph.D.,b and Pari K. Basrur, Ph.D.b Hamilton Health Sciences, Hamilton, and University of Guelph, Guelph, Ontario, Canada
Objective: To determine the cause of infertility in a couple whose oocytes failed to mature in two consecutive fertility treatments. Design: Case report. Setting: University-based IVF program. Patient(s): A 32-year-old woman with unexplained infertility. Intervention(s): Cytogenetic evaluation of oocytes that failed to reach meiotic metaphase II stage of maturation. Main Outcome Measure(s): Observation of oocyte maturity and chromosome composition after fixing and staining with Orcein stain. Result(s): Cytogenetic analysis revealed that the oocytes had successfully resumed meiosis. Germinal vesicle breakdown was also indicated, and chromosomes were at metaphase II stage of development. However, meiotic reduction of those chromosomes failed. Conclusion(s): Infertility in this couple seems to be attributed to the failure of the chromosomes to complete the reduction phase of metaphase II of meiosis. (Fertil Steril威 2002;77:844 –5. ©2002 by American Society for Reproductive Medicine.) Key Words: Cytogenetic, human, maturation, meiosis, oocyte
Received June 1, 2001; revised and accepted October 10, 2001. Reprint requests: Michael S. Neal, M.Sc., Gamete Biology Laboratory, Room 2N48, McMaster University Medical Centre, Hamilton Health Sciences, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada (FAX: 905-528-0623; E-mail: [email protected]). a Hamilton Health Sciences. b Department of Biomedical Sciences, University of Guelph. 0015-0282/02/$22.00 PII S0015-0282(01)03257-5
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Meiosis is initiated during mammalian fetal life, but oocyte maturation is arrested at the diplotene stage of the first meiotic prophase (P1 phase oocyte). This immature or unripened oocyte possesses the double (4N) DNA content because the 46 chromosomes are already replicated before its entry into meiotic prophase (1). At birth, the oocyte presents a nuclear structure known as the germinal vesicle that contains highly extended and decondensed (dictyophase) chromosomes. Meiotic arrest persists until sexual maturity, when selected oocytes resume meiosis at each cycle (2). In preparation for ovulation, the oocytes undergo an intense period of growth during which the regulatory components necessary for the completion of meiosis (maturation) and subsequent events (including fertilization and early cleavage divisions) are stockpiled in the oocyte. The series of events initiated by germinal vesicle breakdown involves recondensation of
chromatin, separation of the homologues, and the reconstitution of the chromosomes into the oocyte and first polar body. The extrusion of the first polar body is thought to be the culmination of events in the production of a mature, fertilizable oocyte that is equipped to support normal early embryonic development (1). Thus, oocyte maturation is an essential prelude to fertilization. We report a case of primary infertility in which two consecutive IVF attempts yielded oocytes with no evidence of polar body extrusion and as a consequence no opportunity for fertilization.
CASE REPORT A 32-year-old patient and her 33-year-old husband came to our clinic with a 3-year history of unexplained infertility. No pregnancy was achieved despite 12 treatment cycles with
clomiphene citrate, followed by 3 cycles of gonadotropin stimulation and IUI. She underwent ovarian stimulation using a long luteal protocol of GnRH agonist and recombinant FSH (1,200 IU in total). On day 12 of stimulation, there were four follicles of ⱖ17 mm in diameter, and the E2 level was 18,350 pm/L. On the basis of this result, 10,000 IU of hCG was administered. Approximately 36 hours later, 27 oocytes were retrieved using transvaginal ultrasound– guided aspiration. The oocytes were cultured in groups of three to four oocytes per double-well culture dish, in medium containing human tubal fluid ⫹ 10% synthetic serum substitute. Semen parameters were in the normal range according to World Health Organization guidelines. The specimen was prepared for insemination using the swim-up technique; 100,000 sperm were added to each culture dish. Fertilization rate was assessed 16 –18 hours after insemination. At this time, there was no sign of in vitro maturation or fertilization in any of the oocytes. The oocytes were left in culture, incubated at 37°C for 24 hours, and reexamined. However, no sign of maturation was observed. During follow-up consultation, our patient wished to attempt a second trial. Intracytoplasmic sperm injection was discussed as an option to maximize fertilization, providing mature oocytes were obtained. The ovarian stimulation protocol was similar to that of the previous cycle, using a total of 975 IU of FSH. On day 14 of stimulation, there were three follicles of ⱖ17 mm in diameter; the E2 level was 13,459 pm/L, and 10,000 IU of hCG was administered. This time, 22 oocytes were collected in the manner described previously. Five hours after collection, the cumulus– oocyte complexes were denuded to assess oocyte maturity. None of the 22 oocytes had extruded the first polar body despite “normal” cumulus expansion. Only one oocyte had a germinal vesicle, whereas the remaining 21 were deemed to be in metaphase I. We were unable to proceed with the intracytoplasmic sperm injection procedure based on these results. The oocytes were left in culture overnight; however, they did not undergo further maturation. As a result, the couple decided to donate these oocytes for research. Signed and informed consent was obtained according to Hamilton Health Sciences policy. Three oocytes were fixed and processed for electron microscopy, which revealed no overt morphological abnormality. The remaining 19 oocytes were fixed and stained for chromosome analysis. Eleven of these yielded analyzable spreads, all of which revealed chromatin in metaphase II. Of
FERTILITY & STERILITY威
these, 9 had two small, closely placed nuclei displaying irregular chromatin condensation, and the remaining 2 had extremely condensed chromatin. The stage at which the meiotic process was arrested in these oocytes was difficult to determine. Ultrastructural details of the three oocytes included cortical granules of varying degrees of condensation and normal mitochondrial maturation, including some giant mitochondria. The microvilli protruding to the perivitelline space were distorted and/or stunted, suggesting that the transportation of metabolites into the oocyte could have been compromised. Chromosome analysis of our patient’s peripheral blood sample revealed an overtly normal female karyotype.
DISCUSSION Approximately 20%–30% of oocytes collected for IVF are meiotically immature at the time of “harvest” from the ovary (1), probably because of the stimulation of multiple follicles. However, it is extremely rare for complete oocyte maturation failure to occur in IVF treatment. This phenomenon has only been reported twice previously (3, 4). Hartshorne et al. (3) attempted unsuccessfully to overcome the failed oocyte maturation by culturing immature oocytes with exogenous gonadotropins. The maturation failure was attributed to the failure of oocytes to enter the metaphase I phase of meiosis. In the case reported here, germinal vesicle breakdown was evident in a majority of oocytes, and meiosis not only had resumed, but the chromatin characteristics indicated transition to metaphase II. It would appear that the cytoplasmic and nuclear processes were interrupted after this stage, leaving the nuclear contents within the ooplasm. The failure to extrude the polar bodies in this case would appear to be due to the inability of the meiotic spindle to form and/or segregate the products of meiosis I. In conclusion, we believe that the fertilization failure and infertility in the present case may be attributable to the uncoupling of nuclear and cytoplasmic maturation processes, leaving the chromosomes in an “unreduced” state. References 1. Avrech OM, Goldman GA, Rufas O, Stein A, Amit S, Yoles I, et al. Treatment variable in relation to oocyte maturation: lessons from a clinical micromanipulation-assisted in vitro fertilization program. J Assist Reprod Genet 1997;14:337– 42. 2. Dekel N. Spatial relationship of follicular cells in the control of meiosis. Prog Clin Biol Res 1998;267:87–101. 3. Hartshorne G, Montgomery S, Klentzeris L. A case of failed oocyte maturation in vivo and in vitro. Fertil Steril 1999;71:567–70. 4. Rudak E, Dor J, Kimchi, M, Goldman B, Levran D, Mashiach S. Anomalies of human oocytes from infertile women undergoing treatment by in vitro fertilization. Fertil Steril 1990;54:292– 6.
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Cytogenetic evaluation of human oocytes that failed to complete meiotic maturation in vitro Michael S. Neal, M.Sc.,a Lisa Cowan, R.T.,a Jackie Pierre Louis, B.Sc.,a Ed Hughes, M.D.,a W. Allan King, Ph.D.,b and Pari K. Basrur, Ph.D.b Hamilton Health Sciences, Hamilton, and University of Guelph, Guelph, Ontario, Canada
Objective: To determine the cause of infertility in a couple whose oocytes failed to mature in two consecutive fertility treatments. Design: Case report. Setting: University-based IVF program. Patient(s): A 32-year-old woman with unexplained infertility. Intervention(s): Cytogenetic evaluation of oocytes that failed to reach meiotic metaphase II stage of maturation. Main Outcome Measure(s): Observation of oocyte maturity and chromosome composition after fixing and staining with Orcein stain. Result(s): Cytogenetic analysis revealed that the oocytes had successfully resumed meiosis. Germinal vesicle breakdown was also indicated, and chromosomes were at metaphase II stage of development. However, meiotic reduction of those chromosomes failed. Conclusion(s): Infertility in this couple seems to be attributed to the failure of the chromosomes to complete the reduction phase of metaphase II of meiosis. (Fertil Steril威 2002;77:844 –5. ©2002 by American Society for Reproductive Medicine.) Key Words: Cytogenetic, human, maturation, meiosis, oocyte
Received June 1, 2001; revised and accepted October 10, 2001. Reprint requests: Michael S. Neal, M.Sc., Gamete Biology Laboratory, Room 2N48, McMaster University Medical Centre, Hamilton Health Sciences, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada (FAX: 905-528-0623; E-mail: [email protected]). a Hamilton Health Sciences. b Department of Biomedical Sciences, University of Guelph. 0015-0282/02/$22.00 PII S0015-0282(01)03257-5
844
Meiosis is initiated during mammalian fetal life, but oocyte maturation is arrested at the diplotene stage of the first meiotic prophase (P1 phase oocyte). This immature or unripened oocyte possesses the double (4N) DNA content because the 46 chromosomes are already replicated before its entry into meiotic prophase (1). At birth, the oocyte presents a nuclear structure known as the germinal vesicle that contains highly extended and decondensed (dictyophase) chromosomes. Meiotic arrest persists until sexual maturity, when selected oocytes resume meiosis at each cycle (2). In preparation for ovulation, the oocytes undergo an intense period of growth during which the regulatory components necessary for the completion of meiosis (maturation) and subsequent events (including fertilization and early cleavage divisions) are stockpiled in the oocyte. The series of events initiated by germinal vesicle breakdown involves recondensation of
chromatin, separation of the homologues, and the reconstitution of the chromosomes into the oocyte and first polar body. The extrusion of the first polar body is thought to be the culmination of events in the production of a mature, fertilizable oocyte that is equipped to support normal early embryonic development (1). Thus, oocyte maturation is an essential prelude to fertilization. We report a case of primary infertility in which two consecutive IVF attempts yielded oocytes with no evidence of polar body extrusion and as a consequence no opportunity for fertilization.
CASE REPORT A 32-year-old patient and her 33-year-old husband came to our clinic with a 3-year history of unexplained infertility. No pregnancy was achieved despite 12 treatment cycles with
clomiphene citrate, followed by 3 cycles of gonadotropin stimulation and IUI. She underwent ovarian stimulation using a long luteal protocol of GnRH agonist and recombinant FSH (1,200 IU in total). On day 12 of stimulation, there were four follicles of ⱖ17 mm in diameter, and the E2 level was 18,350 pm/L. On the basis of this result, 10,000 IU of hCG was administered. Approximately 36 hours later, 27 oocytes were retrieved using transvaginal ultrasound– guided aspiration. The oocytes were cultured in groups of three to four oocytes per double-well culture dish, in medium containing human tubal fluid ⫹ 10% synthetic serum substitute. Semen parameters were in the normal range according to World Health Organization guidelines. The specimen was prepared for insemination using the swim-up technique; 100,000 sperm were added to each culture dish. Fertilization rate was assessed 16 –18 hours after insemination. At this time, there was no sign of in vitro maturation or fertilization in any of the oocytes. The oocytes were left in culture, incubated at 37°C for 24 hours, and reexamined. However, no sign of maturation was observed. During follow-up consultation, our patient wished to attempt a second trial. Intracytoplasmic sperm injection was discussed as an option to maximize fertilization, providing mature oocytes were obtained. The ovarian stimulation protocol was similar to that of the previous cycle, using a total of 975 IU of FSH. On day 14 of stimulation, there were three follicles of ⱖ17 mm in diameter; the E2 level was 13,459 pm/L, and 10,000 IU of hCG was administered. This time, 22 oocytes were collected in the manner described previously. Five hours after collection, the cumulus– oocyte complexes were denuded to assess oocyte maturity. None of the 22 oocytes had extruded the first polar body despite “normal” cumulus expansion. Only one oocyte had a germinal vesicle, whereas the remaining 21 were deemed to be in metaphase I. We were unable to proceed with the intracytoplasmic sperm injection procedure based on these results. The oocytes were left in culture overnight; however, they did not undergo further maturation. As a result, the couple decided to donate these oocytes for research. Signed and informed consent was obtained according to Hamilton Health Sciences policy. Three oocytes were fixed and processed for electron microscopy, which revealed no overt morphological abnormality. The remaining 19 oocytes were fixed and stained for chromosome analysis. Eleven of these yielded analyzable spreads, all of which revealed chromatin in metaphase II. Of
FERTILITY & STERILITY威
these, 9 had two small, closely placed nuclei displaying irregular chromatin condensation, and the remaining 2 had extremely condensed chromatin. The stage at which the meiotic process was arrested in these oocytes was difficult to determine. Ultrastructural details of the three oocytes included cortical granules of varying degrees of condensation and normal mitochondrial maturation, including some giant mitochondria. The microvilli protruding to the perivitelline space were distorted and/or stunted, suggesting that the transportation of metabolites into the oocyte could have been compromised. Chromosome analysis of our patient’s peripheral blood sample revealed an overtly normal female karyotype.
DISCUSSION Approximately 20%–30% of oocytes collected for IVF are meiotically immature at the time of “harvest” from the ovary (1), probably because of the stimulation of multiple follicles. However, it is extremely rare for complete oocyte maturation failure to occur in IVF treatment. This phenomenon has only been reported twice previously (3, 4). Hartshorne et al. (3) attempted unsuccessfully to overcome the failed oocyte maturation by culturing immature oocytes with exogenous gonadotropins. The maturation failure was attributed to the failure of oocytes to enter the metaphase I phase of meiosis. In the case reported here, germinal vesicle breakdown was evident in a majority of oocytes, and meiosis not only had resumed, but the chromatin characteristics indicated transition to metaphase II. It would appear that the cytoplasmic and nuclear processes were interrupted after this stage, leaving the nuclear contents within the ooplasm. The failure to extrude the polar bodies in this case would appear to be due to the inability of the meiotic spindle to form and/or segregate the products of meiosis I. In conclusion, we believe that the fertilization failure and infertility in the present case may be attributable to the uncoupling of nuclear and cytoplasmic maturation processes, leaving the chromosomes in an “unreduced” state. References 1. Avrech OM, Goldman GA, Rufas O, Stein A, Amit S, Yoles I, et al. Treatment variable in relation to oocyte maturation: lessons from a clinical micromanipulation-assisted in vitro fertilization program. J Assist Reprod Genet 1997;14:337– 42. 2. Dekel N. Spatial relationship of follicular cells in the control of meiosis. Prog Clin Biol Res 1998;267:87–101. 3. Hartshorne G, Montgomery S, Klentzeris L. A case of failed oocyte maturation in vivo and in vitro. Fertil Steril 1999;71:567–70. 4. Rudak E, Dor J, Kimchi, M, Goldman B, Levran D, Mashiach S. Anomalies of human oocytes from infertile women undergoing treatment by in vitro fertilization. Fertil Steril 1990;54:292– 6.
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