Mitochondria in oocyte aging: current understanding.

The oocyte is the largest cell found in multicellular organisms. Mitochondria, as the energy factories for cells, are found in high numbers in oocytes, as they provide the energy for oocyte maturation, fertilization, and embryo formation via oxidative phosphorylation. Failure of assisted reproduction is mainly attributed to oocyte aging and increased aneuploidy. As the most numerous organelle in the oocyte, the mitochondrion has been confirmed as a crucial player in the process of oocyte aging, which is highly influenced by mitochondrion dysfunction. Every mitochondrion contains one or more mitochondrial DNA (mtDNA) molecule, which, at about 16.5 KD in length, encodes 13 proteins. In this review, we discuss the function of mitochondria and the relationship between mtDNA and oocyte aging. We also discuss technologies that aim to enhance oocyte developmental potential and delay ovarian aging.

A successful pregnancy following 'double rescue' egg retrieval in a woman with Natural cycle IVF.

A case is reported of a patient with critically low anti-mullerian hormone (AMH) having successful 'rescue' oocyte retrieval in natural in vitro fertilization (IVF) cycle 24 hours after the initial procedure had resulted in no oocyte being collected. Following administration of 10,000 units human chorionic hormone (hCG) a second retrieval was performed 24 hours after the first, producing one Metaphase-I oocyte. After in vitro maturation (IVM) over 4 hours in oocyte maturation medium, introcytoplasmic sperm injection (ICSI) was performed. A subsequent transfer of a 2-cell embryo proceeded on day 2. A positive hCG was recorded 15 days after embryo transfer (ET), and a viable clinical pregnancy has been confirmed. We believe this is the first reported case of a successful egg collection following a prior failed follicle aspiration in Natural IVF cycle. Factors such as good peri-follicular flow and initial follicular fluid cell content are probably essential before attempting a repeat procedure. This report highlights the importance of rescue IVM when an immature oocyte is collected.

Fertility preservation treatment for young women with autoimmune diseases facing treatment with gonadotoxic agents.

OBJECTIVE: To describe a case series of seven women with SLE and other systemic autoimmune rheumatic diseases (SARDs) who required cyclophosphamide therapy and underwent fertility preservation treatments. METHODS: Of the seven patients reported here, five women had SLE with nephritis, the sixth had immune thrombocytopenia purpura (ITP) and the seventh had microscopic polyangiitis (MPA) with renal involvement. All women were nulliparous and younger than 35 yrs. RESULTS: Patients with SLE underwent in vitro maturation (IVM) of immature oocytes aspirated during a natural menstrual cycle followed by vitrification of the matured oocytes if a male partner was not available, or vitrification of embryos if one was available. The patient with ITP and the patient with MPA underwent gonadotropin ovarian stimulation followed by oocyte or embryo vitrification. All women completed fertility preservation treatment successfully and mature oocytes or embryos (36 and 13, respectively) were vitrified. No complications were associated with this treatment and cytotoxic therapy was initiated as scheduled in all cases. CONCLUSIONS: Oocyte or embryo cryopreservation should be considered for fertility preservation in young women with SARDs who face imminent gonadotoxic treatment. In patients, where gonadotropin ovarian stimulation is deemed unsafe, IVM of immature oocytes, aspirated during a natural menstrual cycle, followed by vitrification or fertilization of the mature oocytes, seems to be safe and feasible. For patients in whom hormonal ovarian stimulation is not contraindicated, this method may be considered depending on the urgency to start cytotoxic therapy.

Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report.

We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.

In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience.

BACKGROUND: In vitro oocyte maturation (IVM) permits the use of immature oocytes in IVF. IVM does not require ovarian stimulation and so can be offered to patients at risk of ovarian hyperstimulation syndrome. METHODS: For this indication, we carried out 45 cycles of IVM in 33 women with polycystic ovarian syndrome (PCOS). RESULTS: A total of 509 cumulus-oocyte complexes was obtained; 276 (54.2%) oocytes matured in 24 h and 45 (8.8%) in 48 h. The normal fertilization (2PN) rate of oocytes matured in 24 and 48 h was 69.5 and 73.3% respectively. Among the 214 embryos obtained, 103 were transferred and 30 were frozen. Forty transfers were performed (2.5 embryos/transfer). Eleven women had a positive beta-hCG test (26.2% of pregnancies/puncture, 27.5% of pregnancies/transfer) and nine women had a clinical pregnancy (20.0% of pregnancies/puncture, 22.5% of pregnancies/transfer). Five babies have been born and one pregnancy is ongoing. Results of the clinical examination carried out at birth were normal. CONCLUSIONS: Our results show that IVM may be offered as an alternative to conventional IVF and to ovarian drilling in women with PCOS. The role of IVM in the therapeutic armamentarium for this condition should be further clarified.

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro.

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles.

The present experiments compared the maturational and developmental competence of immature oocytes derived from stimulated cycles, following culture in a newly designed in-vitro maturation medium (IVM-medium) or in standard tissue culture medium (TCM-199; control). The results indicated that maturation and fertilization rates were comparable when the cumulus-free M-I stage oocytes were matured in the IVM-medium (78.6%) or the control medium (70.8%). However, there was a significant difference in blastocyst development (P < 0.05) when M-I oocytes were matured in these two media (19.6 versus 7.7%). Both maturation and early embryonic development rates of GV-stage oocytes were significantly higher (P < 0.01) in the IVM-medium (maturation: 75.7%; blastocyst: 12.9%) compared with control (maturation 55.7%; blastocyst: 0.0%). Moreover, embryos developed to the blastocyst stage at a higher rate in both media if GV-stage oocytes had matured within 24 h compared with 48 h of culture. These results demonstrate that immature human oocytes derived from stimulated ovaries can achieve maturation and early embryonic development in vitro, especially in the new IVM-medium, which may allow additional embryos to be produced for clinical use at embryo transfer.

Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome.

This case report describes the birth of a healthy infant after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from an anovulatory woman with polycystic ovarian syndrome (PCOS). To initiate the treatment cycle, the patient received intravaginal progesterone at night for 10 days to induce a withdrawal bleed. Oocyte retrieval was performed on day 11 following a withdrawal bleed. The patient was administered 10,000 IU of HCG subcutaneously 36 h prior to oocyte collection. A total of 63 immature oocytes were obtained; 10 were morphologically abnormal. Following incubation for 24--48 h in the maturation medium, TC-199 supplemented with 20% patient's own serum, 75 mIU/ml FSH and LH, 77.4% (41/53) of the oocytes were at the metaphase-II stage. Thirty-one (31/41, 75.6%) were fertilized using ICSI with her husband's spermatozoa, 15 fertilized oocytes were cultured for embryo transfer and 16 were frozen at the pronuclear stage. Pregnancy ensued following fresh embryo transfer. Unfortunately, the pregnancy was miscarried eight weeks later. However, the second frozen-thawed embryo transfer attempt resulted in a full-term pregnancy with delivery of a healthy male infant.

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro.

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL(-1) FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen-thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 microM calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (10.8% v 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca(2+)-ionophore. However, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10000g and stimulation with calcium ionophore A23187 than in the control (18.4% v 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be < or = 7000g to enhance the visibility of nuclear elements for further micromanipulation.

Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome.

The present study examined whether the rates of oocyte maturation, fertilization and development, as well as pregnancy rate could be improved by human chorionic gonadotrophin (HCG) priming 36 h before immature oocyte retrieval in patients with polycystic ovarian syndrome (PCOS). Immature oocyte retrieval was performed on day 10-14 of the cycles and patients were randomly allocated either to be primed with 10 000 IU of HCG before the retrieval, or not primed. Immature oocytes were cultured for 24-48 h in TC-199 medium with 20% (v/v) inactivated fetal bovine serum (FBS) supplemented with 75 mIU/ml follicle stimulating hormone (FSH) and luteinizing hormone (LH). Intracytoplasmic sperm injection (ICSI) was performed in all mature oocytes and the resulting embryos were transferred on day 2 or 3 after ICSI. A total of 17 patients underwent 24 completed treatment cycles. Thirteen cycles were primed with HCG and 11 other cycles were not primed. The mean number of oocytes retrieved was comparable in the two groups (7.8 +/- 3.9 versus 7.4 +/- 5.2). The percentage of oocytes achieving maturation at 48 h was significantly higher (P < 0.05) in the HCG-primed group (84.3%, 86/102) than in the non-HCG-primed group (69.1%, 56/81). Oocyte maturation was hastened in the HCG-primed group. Following 24 h of culture, 78.2 +/- 7.1% of oocytes were matured in the HCG-primed group compared with 4.9 +/- 2.5% of oocytes in the non-HCG-primed group (P < 0.001). There were no significant differences in the rates of oocyte fertilization and cleavage in these two groups. There were five clinical pregnancies (38.5%) in the HCG-primed group, and three pregnancies (27.3%) in the non-HCG-primed group.

Pregnancies resulting from in vitro matured oocytes retrieved from patients with polycystic ovary syndrome after priming with human chorionic gonadotropin.

OBJECTIVE: To describe pregnancies that resulted from in vitro matured oocytes derived from two unstimulated, anovulatory patients with polycystic ovary syndrome after priming with hCG before oocyte retrieval. DESIGN: Case series. SETTING: McGill Reproductive Center, Royal Victoria Hospital, McGill University. PATIENT(S): Two women with polycystic ovary syndrome. INTERVENTION(S): Progesterone induction of a withdrawal bleeding episode. The administration of subcutaneous hCG 36 hours before oocyte retrieval in a natural menstrual cycle. In vitro maturation of immature oocytes, fertilization, and ET. Luteal support with oral estrogen and progesterone. MAIN OUTCOME MEASURE(S): Pregnancy outcome. RESULT(S): Two clinical pregnancies were confirmed after oocyte maturation in vitro, fertilization with intracytoplasmic sperm injection, and ET. CONCLUSION(S): The administration of hCG 36 hours before harvesting of immature oocytes may improve the maturational and developmental competence of the oocytes and the pregnancy rates of unstimulated patients with polycystic ovary syndrome.

Protein phosphorylation in bovine oocytes following fertilisation and parthenogenetic activation in vitro.

This study examined the event of protein phosphorylation in bovine oocytes in response to sperm penetration and parthenogenetic activation. In vitro matured oocytes were labelled with [32P]orthophosphate at 3 h intervals from 3 h to 18 h or from 0 h to 12 h following in vitro fertilisation and parthenogenetic activation, respectively. The level of protein dephosphorylation, at approximately 43 kDa, was similar in fertilised and parthenogenetically activated bovine oocytes. However, the level of protein phosphorylation at 40 kDa, 23 kDa and 18 kDa was different between these two samples. There were no such changes of protein phosphorylation and dephosphorylation in the control oocytes. Further, by two-dimensional gel electrophoresis there is a difference in the level of protein phosphorylation at 18 kDa between the fertilised and activated oocytes. These results suggest that this protein phosphorylation may be related to the formation of the male pronucleus in bovine oocytes.

Production of steroids from human cumulus cells treated with different concentrations of gonadotropins during culture in vitro.

OBJECTIVE: To evaluate the output of E2 and progesterone produced by cumulus cells, derived from mature and immature oocytes, in culture medium. DESIGN: Prospective randomized study. SETTING: McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. PATIENT(S): Twenty-one women, <38 years of age and with normal menstrual cycles, who were undergoing intracytoplasmic sperm injection for assisted reproduction. INTERVENTION(S): Culture medium with or without fetal bovine serum (FBS) supplemented with either a physiologic (75 mIU/mL) or a supraphysiologic (7,500 mIU/mL) concentration of gonadotropins. MAIN OUTCOME MEASURE(S): Comparison of steroid levels in culture medium. RESULT(S): Estradiol secretion was significantly increased in the culture medium with FBS supplemented with both concentrations of FSH alone compared with control. However, E2 secretion was inhibited by both concentrations of FSH with LH. The level of E2 was undetectable in the medium without FBS even after supplementation with both concentrations of FSH alone, hCG alone, and FSH with LH. Progesterone production was increased in the medium with FBS supplemented with FSH alone, hCG alone, and FSH with LH compared with control. There was no difference in progesterone levels in the culture medium without FBS supplemented with both concentrations of FSH alone and hCG alone compared with control. However, progesterone secretion was increased in the medium without FBS supplemented with a physiologic concentration of FSH with LH. CONCLUSION(S): Culture medium with FBS supplemented with a physiologic and a supraphysiologic concentration of FSH stimulates E2 secretion from cumulus cells derived from mature and immature oocytes. This suggests that it may be not necessary to add E2 to the culture medium for maturation in vitro of immature human oocytes retrieved from patients undergoing stimulated cycles.

Maturation in vitro of immature human oocytes for clinical use.

Human oocyte maturation is considered as the reinitiation and completion of the first meiotic division from the germinal vesicle stage (prophase I) to metaphase II, and the accompanying cytoplasmic maturation for fertilization and early embryonic development. Immature human oocytes obtained from patients undergoing gynaecological surgery, or ovulation induction or having polycystic ovary syndrome (PCOS) can be matured and fertilized in vitro. To date, 80% of immature oocytes matured to metaphase II when cultured in maturation medium supplemented with gonadotrophins and 85% of matured oocytes fertilized and cleaved in vitro. Following transfer of these embryos, pregnancies and live births have been achieved. However, the capacity for oocyte maturation was different when the immature oocytes were retrieved from PCOS patients and when the oocytes were cryopreserved at germinal vesicle stage.

Effect of progesterone and/or estradiol-17beta on sperm penetration in vitro of bovine oocytes.

The effect of progesterone (P4) and estradiol-17beta (E2) on sperm penetration was evaluated by in vitro fertilization technique. When spermatozoa were treated with modified Tyrode's medium (mTALP) alone, mTALP + 1.0 microg/ml heparin (H), mTALP + 0.1 microg/ml P4, mTALP + 0.1 microg/ml E2, or mTALP + 0.1 microg/ml P4 + 0. 1 microg/ml E2, the percentages of penetrated oocytes were 11% (8/74), 94% (54/60), 19% (12/64), 10% (6/63) and 13% (8/62), respectively. The penetration rates by spermatozoa treated with H, H + P4, H + E2 and H + P4 + E2 were 94% (118/125), 100% (138/138), 95% (129/136), and 94% (100/106), respectively. However, the oocyte penetration rates significantly increased (P < 0.01) 4, 5 and 6 h after insemination, respectively, when the spermatozoa were treated with H + P4, H + E2 and H + P4 + E2 compared with that of the control (H). Cleavage rates also increased significantly (P < 0.01) 24 and 30 h following insemination, respectively, when spermatozoa were treated with H + P4, H + E2 and H + P4 + E2 compared with that of H. Nevertheless, There was no difference in the production of > or = 32 cell stage embryos among the 4 treatments (19% = 28/149, 18% = 32/176, 18% = 23/128 and 22% = 26/120, respectively). These results indicate that the time course of capacitated sperm penetration was accelerated by progesterone and estradiol-17beta but it did not affect subsequent early embryonic development.

Protein synthesis is not required for male pronuclear formation in bovine zygotes.

Following fertilisation, the sperm triggers a series of intracellular changes which initiate oocyte activation and pronuclear formation. Oocyte activation can also be induced artificially by several chemicals, such as the calcium ionophore A23187. The sperm nucleus is transformed into the male pronucleus through the interaction of oocyte cytoplasmic factors. The profile of protein synthesis is different in bovine oocytes following fertilisation and parthenogenetic activation. The formation of male and female pronuclei was not blocked by the presence of the protein synthesis inhibitor cycloheximide. These results suggest that bovine oocyte activation by sperm and parthenogenetic activation induce different cytoplasmic responses for protein synthesis and that new protein synthesis is not required for male pronuclear formation in bovine zygotes.