Developmental competence of human embryos derived from in vitro maturation of immature oocytes retrieved during cesarean section

bs_bs_banner doi:10.1111/jog.12186 J. Obstet. Gynaecol. Res. Vol. 40, No. 2: 459 464, February 2014 Developmental competence of human embryos derived from in vitro maturation of immature oocytes retrieved during cesarean section Pallop Pongsuthirak 1 and Teraporn Vutyavanich 2 1 Buddhachinaraj Hospital Medical School, Phitsanulok, and 2 Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Abstract Aim: To investigate the developmental competence of human embryos that originated from in vitro matured oocytes retrieved during cesarean section. Methods: Immature oocytes were collected from 95 pregnant women, who underwent cesarean section at Buddhachinaraj Hospital Medical School and consented to participate in the study. Retrieved oocytes were cultured in blastocyst medium supplemented with 75 IU/L of human menopausal gonadotropin. Oocyte maturation was assessed at 30 and 48 h after culture. In vitro matured oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 h. The fertilization, cleavage and blastocyst formation rates were observed. Results: Maturation rate of oocytes after 30 h of culture was 67.9% compared with 13.1% at 48 h (P < 0.0001). Insemination of oocytes in both groups resulted in similar fertilization, cleavage and blastocyst formation rates. Conclusion: A large proportion of oocytes retrieved at the time of cesarean section exhibited the capacity to undergo maturation in vitro. They can be fertilized and developed into good-quality blastocyst stage. Key words: cesarean section, cytoplasmic maturation, in vitro maturation, nuclear maturation. Introduction In vitro fertilization (IVF) is a very successful treatment for infertile couples. However, there are some disadvantages to this procedure: the high costs and inconvenience of gonadotropin administration, medication side-effects, multiple clinic visits, and the risk of ovarian hyperstimulation syndrome and ovarian cancer. 1,2 In vitro maturation (IVM) of immature oocytes retrieved from unstimulated cycle and their use in IVF is an attractive alternative to overcome the disadvantages of standard IVF. Human oocytes remain arrested in the first meiotic prophase until a process of recruitment initiates their growth. A single follicle achieves dominance and the others undergo atresia during follicular growth. When the follicles reach a certain size, the enclosed oocytes are capable of resuming and completing meiosis once they are released from the follicles. 3 In vitro maturation of human oocytes is an emerging technology, with rapidly expanding clinical uses. In 1935, Pincus and Enzmann reported that immature rabbit oocytes removed from their natural ovarian environment could undergo spontaneous maturation Received: December 11 2012. Accepted: May 13 2013. Reprint request to: Dr Pallop Pongsuthirak, Department of Obstetrics and Gynecology, Buddhachinaraj Hospital Medical School, Srithummatripidok Rd. Aumphur Muang, Phitsanulok 65000, Thailand. Email: palloppong@yahoo.co.th Conflict of interest: The authors declare no conflict of interest. There are no financial or personal relationships with other people or organizations that could inappropriately influence the results of the study. 2013 The Authors 459

P. Pongsuthirak and T. Vutyavanich and achieve fertilization. 4 Similar results were reported in humans by Edwards in 1965. 5 The research continued thereafter and demonstrated that IVM is a successful treatment for women with polycystic ovaries, with or without follicular priming. The indications for IVM currently expand to include oocyte donation, poor responders and fertility preservation before chemotherapy or radiotherapy. However, the sources of available immature oocytes for IVM are largely nonpregnant women. Interestingly, there are many preantral follicles in the ovaries of pregnant women, and some studies have reported pregnancies from IVM of oocytes retrieved during cesarean section. 6 8 However, there is still no data on a large pool of immature oocytes that are obtained during pregnancy. The purpose of this study was, therefore, to determine the rates of maturation and fertilization of a large cohort of immature oocytes obtained during cesarean section, and to observe their subsequent embryo developments. Methods Patients The research ethics committee of Buddhachinaraj Hospital Medical School approved this study. Pregnant women, who underwent cesarean section at the hospital, were invited to participate in the study if they met the following inclusion criteria: (i) aged 18 40 years; (ii) singleton pregnancy with a gestational age of 37 41 weeks; (iii) elective repeated or emergency cesarean section not due to fetal distress; (iv) gave their written informed consent to participate in the study; and (v) had no chronic diseases or medical or obstetric complications that could put them in jeopardy by prolonging either anesthesia or surgery. Patients were excluded if they: (i) could not read or write or understand the Thai language; and (ii) had excessive blood loss or unstable vital signs during the operation. Oocyte collection, maturation, fertilization and embryo development Cesarean section was performed under general anesthesia or spinal block. After delivery of the fetus and closure of the uterine wound, all visible follicles were aspirated with a 22-G needle connected to a 5-mL syringe, filled with 1 ml of warmed flushing medium (Ferticult Flushing). Aspirated fluid was decanted into a sterile 35 mm 10 mm tissue culture dish, and examined using a dissecting microscope. Oocytes with signs of atresia (dark or shrunken, irregular cytoplasm), nude cumulus or mechanical damage were discarded. Healthy appearing oocytes (with multilayered or sparse cumulus) were cultured in groups of three in 25-mL drops of blastocyst medium supplemented with 75 IU/L of human menopausal gonadotropin (IVF-M) under paraffin oil at 37 C in a humidified atmosphere of 6% CO 2 in air. The maturity of the oocytes was determined using an inverted microscope at 30 and 48 h post-culture. Oocyte handling procedures were conducted on warm stages and plates at 37 C. Before maturity assessment, oocytes were denuded of their cumulus cells by a 30-s exposure to 80 IU hyaluronidase in flushing medium, and the use of a denuding pipette (Flexipet). Oocytes with extruded first polar bodies were inseminated by intracytoplasmic sperm injection (ICSI). Spermatozoa for ICSI were prepared by Sil-select Plus gradient centrifugation at 300 g for 20 min. The sperm pellet was washed twice with 2 ml of flushing medium at 200 g. Following ICSI, oocytes were cultured in groups of up to five in 20-mL drops of cleavage medium in a tissue culture dish covered with paraffin oil. Oocytes that remained immature at the first examination were cultured for another 18 h, and rechecked for maturation. ICSI was then performed on late-matured oocytes. Fertilization was assessed 18 h after ICSI for the appearance of two pronuclei and two polar bodies. After 72 h of culture in cleavage medium, the embryos were transferred into blastocyst medium, and cultured for another 72 h. Embryos were examined once every 24 h. The stage of embryo development was determined on the basis of the number of blastomeres. Only fully expanded blastocysts and hatching or hatched blastocysts, containing a distinct inner cell mass and trophectoderm layer, were considered to be goodquality blastocysts. Statistical analysis Stata ver. 11 software was used for all statistical analyses. Comparisons of frequency data between groups, such as fertilization and cleavage rates, were performed using c 2 -tests. A P-value below 0.05 was considered to indicate statistical significance. Results Ninety-five pregnant women at 37 41 weeks of gestation, who underwent cesarean section between July 2011 and March 2012, were enrolled into the study. Their mean age, gravidity, parity, body mass index, 460 2013 The Authors

Embryo competence after IVM infant birthweight and placental weight are shown in Table 1. The average oocyte collection time was 10.9 3.8 min (range, 4 28). A total of 1227 oocytes were retrieved (mean, 12.9 6.5 oocytes/subject; range, 1 37). The number of oocytes with degenerative appearance at the time of recovery was 179 (14.6%) and the number of denuded oocytes was nine (0.7%). A total of 1039 healthy oocytes with cumulus complexes were included in the study. The majority of oocytes (83.8%) that would undergo maturation extruded the first polar bodies within 30 h of in vitro culture. Sixteen out of 842 in vitro matured oocytes (1.9%) had abnormally large polar bodies, and none of them fertilized after ICSI. There was no significant difference in the fertilization rates, cleavage rates, and rates of good-quality blastocysts in oocytes that matured at 30 or 48 h of culture (Table 2). Discussion During pregnancy, oocytes are exposed to high circulating levels of estrogen, progesterone and hcg. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from the pituitary gland are suppressed. Despite a relatively FSH-deficient milieu, many follicles can still develop into small antral follicles (4 5 mm in diameter) but they do not reach the pre-ovulatory stage. 9,10 High progesterone milieu Table 1 Characteristics of 95 pregnant women who participated in the study Characteristics Mean SD Range Age (years) 27.0 6.0 18 40 Gravidity 1.5 0.8 1 5 Parity 0.8 0.6 0 3 Gestational age (weeks) 38.4 0.9 37 41 Body mass index (kg/m 2 ) 28.0 4.8 20.8 49.6 Infant birthweight (g) 3113.7 360.7 2380 4060 Placental weight (g) 661.6 108.8 500 880 SD, standard deviation. during the luteal phase and pregnancy was previously believed to exert a negative effect on the developmental capacity of the oocytes. This concept is now challenged by new findings that antral follicular growth may start and continue in any phase of the menstrual cycle, and even during pregnancy. 11 13 As such, some of the antral follicles may be in an earlier phase of follicular growth, and not necessarily be at the atretic stage of development. 11 13 As demonstrated in our study, immature oocytes with intact cumulus mass from nonatretic follicles can indeed undergo nuclear maturation at a high rate. In this study, an average of 11 (1039/95) immature oocytes of good quality could be recovered per patient during cesarean section. Only 14.6% of oocytes were degenerated and 0.7% was denuded at the time of recovery. The percentage of degenerative oocytes was compatible with a previous report, 7 but that of denuded oocytes was lower, possibly due to the use of a lower aspiration pressure during retrieval in our study. Our results were in line with a previous study, which showed that lower aspiration pressure improved the developmental competence of immature oocytes following IVM and IVF. 14 In vitro maturation of human oocytes requires both nuclear and cytoplasmic maturation. 15 17 Nuclear maturation involves germinal vesicle breakdown, chromosome condensation and segregation, organization of microtubules and the release of the first polar body. After nuclear maturation, the oocytes are arrested again at metaphase II (MII) until fertilization. Cytoplasmic maturation involves organelle reorganization, cytoskeleton dynamics and molecular maturation during oocyte growth and meiosis. 18 Signal transduction between the oocytes and cumulus cells is essential for proper cytoplasmic maturation. 19 Although nude oocytes may undergo polar body extrusion and become MII oocytes, their future development and implantation potential may be impaired. 15 17,19 Thus, denuded immature oocytes were not included in our study. Table 2 Comparison of the developmental capacity of embryos following in vitro maturation for 30 and 48 h Variables 30 h 48 h Total No. of metaphase II oocytes (%) 706 (67.9%) 136 (13.6%) 842 (81.0%)* No. of fertilizations (%) 582 (82.4%) 108 (79.4%) 690 (81.9%) No. of cleavages (%) 493 (84.7%) 85 (78.7%) 578 (83.7%) No. of blastocysts (%) 322 (55.4%) 55 (50.9%) 377 (54.6%) *P < 0.0001. 2013 The Authors 461

P. Pongsuthirak and T. Vutyavanich In vitro maturation is enhanced by the presence of hormones, energy substrates and protein sources. Other factors, such as growth factors, lipids, glycoproteins, steroid hormones and cytokines, may also be involved. Patient-inactivated serum or follicular fluid has been used successfully as exogenous protein sources, but their clinical use is limited due to potential infectious contamination. Commercial media are widely used in clinical IVM but some, such as Medicult IVM medium, also require supplementation with the patient s serum before use. In this study, we used blastocyst medium, as it contained several components necessary for oocyte maturation, namely, glucose, pyruvate, salts, amino acids, vitamins and proteins. 20 Similar to other IVM media, supplementation with human menopausal gonadotropin (FSH and LH 75 IU/L) is required before use. We achieved a maturation rate of 81%, which was compatible with other previous reports. 6 8,20,21 The ability of oocytes to resume meiosis and complete maturation is dependent on follicular size. 22,23 Oocytes from smaller follicles (3 4 mm) had a lower maturation rate than those from larger follicles (9 15 mm). At the time of immature oocyte collection, both ovaries often contain follicles of different sizes. It is, therefore, inevitable that retrieved oocytes show asynchronous maturation during in vitro culture. In this study, we did not record the size of individual follicles at the time of collection, and were unable to correlate the maturation rate with follicular size. Some previous reports on IVM allowed an interval of 48 56 h between the start of in vitro culture and insemination. 24,25 In our study, this period was shortened to 30 h, with satisfactory yields of oocyte maturation (67.9%), fertilization rate (82.4%) and embryo cleavage rate (84.7%). Our results were comparable with other studies in polycystic ovary syndrome patients, which reported a maturation rate of 44 70% after 28 30 h of in vitro culture, an ICSI fertilization rate of 69 78%, and further cleavage rate of 56 82.5%. 26 28 We believed the use of early insemination might prevent oocytes from senescence before insemination, as the optimal insemination time is 2 4 h after extrusion of the first polar body. 17 Leaving the oocytes in MII stage longer than necessary could place them well past the optimal fertilization time and compromise their developmental competence. Previous studies 7,16,17 demonstrated a reduced developmental competence of embryos that were derived from IVM oocytes. These embryos had a high incidence of cleavage arrest at the two- to 16-cell stage in a conventional culture system. Extended culture to the blastocyst is a better way to demonstrate the developmental competence of embryos. In this study, 54.6% of fertilized oocytes developed into good-quality blastocysts, which is within the range observed in embryos (<55%) that were derived from mature oocytes at the time of retrieval. 29 In recent years, the demand for oocyte donation is increasing but the availability of donors is limited. Oocyte donation usually involves ovarian stimulation with gonadotropins to produce multiple oocytes, and retrieving them through a surgical procedure that the donor will not otherwise need. The technology is well established and associated with a low risk for complication. However, ovarian hyperstimulation syndrome remains a life-threatening risk, and there may be other unknown long-term risks, such as ovarian cancer. 30,31 Immature oocyte retrieval at the time of cesarean section is an attractive alternative to conventional oocyte donation. The procedure exposes the donor to minimal additional risk and adds only 10 min or so to the operative time, as shown in our study. Such a procedure provides an access to a large pool of potential oocyte donors of proven fertility, and it may enhance participation in oocyte donation. Identification of pregnant women who have had a previous cesarean section, and are willing to donate their oocytes, would simplify the synchronization of recipients by maintaining them on artificial cycles with hormone replacement in anticipation of the expected date of a repeat cesarean delivery. Collection of immature oocytes during a cesarean section is also a promising option for pregnant women who require fertility preservation, such as those with renal transplantation 32 or those who are pregnant from an IVF treatment and desire future treatment but do not have any surplus frozen embryos. 7 To date, there have been few reports on IVM of immature oocytes collected during the luteal phase and during pregnancy. One study found that these oocytes had a significantly higher maturation rates than those retrieved during the follicular phase, 33 while another study found slightly lower maturation rates but a trend towards a higher fertilization rate. 34 Yet another study found no difference in the efficiency of oocyte retrieval or the outcomes in terms of maturation rates at 24 and 48 h and fertilization rate. 35 In the bovine model, maturation and fertilization rates were shown to be equal in oocytes obtained during the follicular and luteal phase. 36 Despite the conflicting results, all studies including ours concurred that luteal phase IVM 462 2013 The Authors

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