Follicle stimulating hormone effects on immature human oocytes: In vitro maturation and hormone production

Purpose: Our purpose was (1) to determine if in vitro maturation of unstimulated oocytes could be improved with the addition of urofollitropin; (2) to evaluate the output of estradiol, testosterone, progesterone, and androstenedione by the cultured oocyte-cumulus complex; and (3) to ascertain if steroid hormone production of the oocyte-cumulus complex correlates with final oocyte maturation stage. Methods: Fifty-eight immature oocytes were obtained from 11 regularly cycling women undergoing oophorectomy. The oocyte-cumulus complexes were randomly assigned to control medium (Ham’s F-10 supplemented with 7.5% fetal bovine serum) or test medium (control medium supplemented with 75 mIU/ml of urofollitropin). Results: (1) The addition of urofollitropin to oocyte culture medium does not significantly increase the ability of the oocyte to achieve the metaphase II stage; (2) the addition of urofollitropin significantly increases the production of progesterone, testosterone, and androstenedione by the oocyte-cumulus complex; and (3) there is no difference in the production of estradiol, progesterone, testosterone, and androstenedione by the oocyte-cumulus complex at the germinal vesicle, metaphase I or metaphase II stage of oocyte maturation. Conclusions: This information is of importance in the use of oophorectomy specimens for patients who must undergo an oophorectomy but desire to attempt pregnancy using their oocytes, in the use of oophorectomy specimens for donor oocytes, or for patients undergoing in vitro fertilization using immature oocyte collection.     

Isolation,in vitro maturation,and fertilization of germinal vesicle oocytes obtained from the intact murine ovary

The purpose of this investigation was to attempt to develop a process, utilizing a murine model, which would allow more efficient harvesting from the intact ovary and maturation in vitro of germinal vesicle (GV) oocytes. The recovery process yielded 25.5±4.5 ( ±SE) cumulusfree GV oocytes per animal. Treatment groups included culture medium (CM) supplemented with either estradiol (E₂), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), or prolactin (PRL). Among the hormone-free controls 83.2±1.6% of oocytes underwent GV breakdown, whereas 25.3±2.6% developed to the first polar body stage (PB-1) following 18 hr of incubation (n=29 trials). Oocytes progressing to the PB-1 stage were inseminated in vitro. In vitro fertilization (IVF) of pooled in vitro matured (IVM) PB-1 oocytes (judged by two-cell formation) was 19.9%, which was significantly lower than in the group of in vivo matured oocytes (74.7%). E₂ significantly increased the percentage of GV breakdown (control, 76.8±2.5%; E₂ at 10 ng/ml, 92.9±2.5%,P<0.001; E₂ at 100 ng/ml, 93.7±2.1%,P<0.001; and E₂ at 1 g/ml, 86.7±3.3%,P<0.05) but not PB-1 formation. Neither FSH nor hCG significantly increased GV breakdown or PB-1 formation. Prolactin treatment resulted in an increased percentage of PB-1 formation (control, 25.3±2.5%; PRL at 2 g/ml, 35.0±2.9%; and PRL at 20 g/ml, 34.1±1.9%;P<0.01), fertilization (control, 15.3±5.1%; PRL, 33.6±8.5;P<0.01), and subsequent development (control, 3.5±2.3%; PRL, 18.8±5.6%;P<0.01). We conclude that recovery and IVM of GV oocytes is feasible, however, further work is necessary to define optimal conditions.     

In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles

Objective: To evaluate whether germinal vesicle (GV) oocytes from stimulated cycles can be a source of embryos.

Design: In vitro model study.

Setting: Specialized laboratory of women’s clinic.

Patient(s): Women in whom oocytes were retrieved at the GV stage.

Intervention(s): After culture of GV oocytes in the modified TLP medium with human follicular fluid, oocytes that reached the metaphase II stage underwent ICSI. Potential of fertilization and subsequent cleavage of in vitro-matured oocytes was compared with that of an in vivo-matured control.

Main Outcome Measure(s): Maturation rate, rate of pronuclei formation, and developmental activity.

Result(s): The maturation rate of GV oocytes from follicles primed with gonadotropin was not affected by the presence or absence of cumulus. However, the maturation was more synchronous in oocytes with cumulus than in those without cumulus. Proportions of oocytes with two pronuclei and embryos cleaved to the 16-cell stage after ICSI were significantly lower in the oocytes matured in vitro than in the oocytes matured in vivo.

Conclusion(s): Human GV oocytes from stimulated ovaries can be matured, fertilized, and developed in vitro. Production of embryos from GV oocytes will increase the opportunity for achieving pregnancy.     

Treatment variables in relation to oocyte maturation: Lessons from a clinical micromanipulation-assisted in vitro fertilization program

Objective: In an effort to understand the mechanism underlying the improved pregnancy rate observed in IVF cycles when gonadotropin-releasing hormone analogues (GnRH-a) are applied, we investigated a possible relationship between treatment variables and oocyte-nuclear maturity. Design: Nuclear maturity was retrospectively assessed in cumulus-free, denuded oocytes, obtained from women undergoing micromanipulation-assisted IVF treatment following controlled ovarian hyperstimulation with GnRH-a and menotropins. Setting: The setting was the infertility and IVF unit of a tertiary academic medical center. Participants: Two hundred twenty-one patients underwent 435 treatment cycles. Main Outcome Measure: This was the proportion of germinal vesicle-intact immature (GVII) oocytes. Results: One hundred fifty-four of the 3520 oocytes studied (4.4%) were in the GVII stage. These oocytes were found in 66 of the treatment cycles (15.2%) and in 54 of the patients (24.4%). Cycles in which GVII oocytes were detected did not differ from those in which all the aspirated oocytes were mature in the following respects: patient age, type and duration of infertility, controlled ovarian hyperstimulation protocol and time of ovum pickup. However, the GVII group was characterized by a significantly higher peak estradiol level, as well as a higher number of mature follicles visualized sonographically (diameter, >14 mm) and oocytes retrieved. Conclusions: Comparing the present findings with previously published data, it appears that the inclusion of GnRH-a in the stimulation regimen is associated with a lower proportion of immature oocytes. A higher occurrence of oocyte-nuclear immaturity is apparently associated with a significantly better ovarian response to stimulation. The high incidence of immature oocytes observed in patients with normospermic partners and low fertilization rates in previous cycles may suggest that the fertilization failure in some of these cases is due to oocyte, rather than sperm, dysfunction.     

Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles

OBJECTIVE: To describe pregnancies that resulted from in vitro matured oocytes derived from stimulated IVF cycles before cancellation owing to poor response of gonadotropins. DESIGN: Case report.University hospital. PATIENT(S): Eight patients who underwent in vitro maturation.Immature oocyte retrieval, in vitro maturation of immature oocytes, fertilization, and ET. Luteal support with progesterone and plvyeron was given. MAIN OUTCOME MEASURE(S): Pregnancy and live birth. RESULT(S): Three pregnancies (two live births and another ongoing) were achieved after immature oocyte retrieval, in vitro maturation, fertilization with ICSI, and ET. CONCLUSION(S): Immature oocyte retrieval from poor responders during stimulation, followed by in vitro maturation, may be an alternative before the cycle is canceled.     

Full physiological maturation in vitro of immature mouse oocytes induced by sequential treatment with follicle-stimulating hormone and luteinizing hormone

Cumulus cell-enclosed immature mouse oocytes were matured in medium supplemented with various combinations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol. FSH or LH alone stimulated oocyte maturation, resulting in a significant increase in the rate of development to blastocysts following fertilization in vitro and embryo culture. There was no significant difference between FSH and LH. The effect of FSH was neutralized by FSH antiserum, while that of LH was not indicating that the stimulation of maturation by LH was not due to FSH contamination in the LH preparation. When LH was added after 2 hr of culture with FSH (sequential combination), blastocyst development was significantly increased compared with FSH alone, reaching the same level as the in vivo matured oocytes. The addition of estradiol, 0.1 ng/ml to the sequential combination of FSH and LH had no effect, while 0.01 and 1 ng/ml produced a negative effect. The birth rate of normal live offspring following embryo transfer showed no significant difference between embryos derived from oocytes matured in vivo and in vitro (sequential combination with or without 0.1 ng/ml estradiol) or between the two in vitro treatment groups.     

Effects of estradiol on in vitro maturation of buffalo and goat oocytes

PurposeThe effects of estradiol on oocyte development seem to be varied among species. The present study investigated the effects of 17β‐estradiol on in vitro maturation of buffalo and goat oocytes.MethodsCumulus oocyte complexes (COCs) were aspirated from large antral follicles of slaughtered buffalo and goat ovaries. COCs were cultured in TCM‐199 medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17β‐estradiol for in vitro maturation. Then, oocytes were used for the examination of state of nuclear maturation and cumulus expansion.ResultsIn both species, oocytes treated with 17β‐estradiol showed higher cumulus expansion rate than control (0 µg/mL treated). In buffalo, the percentage of oocytes matured to the metaphase II (MII) stage increased in the concentration‐dependent manner of 17β‐estradiol. Similarly, estradiol positively influenced nuclear maturation of goat oocytes in vitro.ConclusionsEstradiol has promoting effects on normalprogress of in vitro oocyte meiosis in buffalos and goats.     

多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

目的评价未成熟卵母细胞体外成熟（IVM）后形成的卵裂期胚胎经慢速冷冻一解冻后的发育能力。方法将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征（PCOS）合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组：复苏胚胎来源于体外成熟的卵母细胞组（IVM组,46例）和复苏胚胎来源于常规体内成熟的卵母细胞组（IVF组,339例）。采用慢冻速溶法解冻移植后比较两组患者的临床结局。结果IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66．67％;IVF组复苏胚胎1605枚,复苏后存活1082枚,复苏率为67．41％,两组比较,差异无统计学意义（P〉0．05）。IVM组患者的临床妊娠率和着床率分别为19．30％（11／57）和10．61％（14／132）,明显低于IvF组临床妊娠率（45．45％,175／385）和着床率（26．14％,240／918;P均〈O．05）。结论体外成熟卵母细胞发育形成的卵裂期胚胎慢速冷冻后临床结局欠佳,可能与冻融前胚胎自身的发育潜力有关。     

In vitro maturation of baboon oocytes retrieved at the time of cesarean section

Objective: To investigate the feasibility of oocyte retrieval at the time of cesarean delivery and the potential of such oocytes to undergo nuclear maturation in vitro using a baboon model and an established culture system.

Design: Randomized, controlled animal study.

Setting: Research foundation and university research laboratory.

Animal(s): Mature pregnant baboons.

Intervention(s): In vitro culture of aspirated oocytes with or without epidermal growth factor (EGF).

Main Outcome Measure(s): Oocyte yield, germinal vesicle breakdown, polar body extrusion.

Result(s): A total of 246 oocytes were retrieved (mean, 35; range, 14–67). Eighty-seven oocytes (35%) underwent germinal vesicle breakdown and 72 oocytes (29%) extruded a polar body. A χ² analysis revealed no significant effect of EGF on outcome parameters. No effect of gestational age or maternal age on oocyte yield or development was observed.

Conclusion(s): A sizeable proportion of oocytes obtained from puerperal primates exhibited the capacity to undergo nuclear maturation in vitro.     

In vitro DNA fluorescence after in vitro fertilization (IVF) failure

Purpose A pilot study was performed to test the diagnostic value of in vitro DNA fluorescence in oocytes that failed to fertilize after IVF. Ten patients with a cleavage rate less than 20% after IVF were included.Results Uncleaved oocytes were observed by fluorescence microscopy after incubation with the DNA fluorescent dye Hoechst 33342. Four main causes which may have contributed to the low cleavage rate were found: (1) sperm incapacity to penetrate the oocyte despite the absence of the usual criteria for male infertility, (2) oocyte immaturity, (3) delayed fertilization, and (4) oocyte abnormalities revealed by aberrations in the morphology of the female chromatin.Conclusions The possibility of a rapid and detailed analysis of the maturational status of unfertilized oocytes, the morphology of the female chromatin, the presence and quantity of spermatozoa tightly bound to the zona pellucida, and sperm penetration into the oocyte without subsequent pronucleus formation, using DNA fluorescence, allows us to clarify further the cause of fertilization failure and to orient infertility treatment toward the male, the female, or both partners.     

Commercially available enhanced in vitro maturation medium does not improve maturation of germinal vesicle and metaphase I oocytes in standard in vitro fertilization cases

Improvements in in vitro maturation techniques have focused on culture optimization to increase oocyte maturation rates. Specialized culture media, now commercially available, did not perform significantly better than standard IVF culture media for maturation of immature oocytes in our normal IVF cases.     

A novel system for in vitro maturation of human oocytes

Objective: To compare in vitro maturation of cumulus-free oocytes in glucose-free medium (P1) and standard medium (TC199).

Design: Prospective, cohort study.

Setting: Assisted reproductive technology program.

Patient(s): One hundred eight patients undergoing ICSI.

Intervention(s): Germinal vesicle–stage or metaphase I–stage oocytes were allocated to culture with P1 or TC199. Metaphase II oocytes were fixed for immunofluorescence analysis or fluorescence in situ hybridization at 24 or 48 hours (or both). Media were compared by performing conditional logistic regression analysis that controlled for egg-specific factors.

Main Outcome Measure(s): Proportion of mature oocytes and appearance of normal spindle-chromosome cytoarchitecture.

Result(s): At 24 hours, more P1 oocytes than TC199 oocytes reached metaphase II (59.7% vs. 44.9%). At 48 hours, 71.7% of P1 oocytes and 61.0% of TC199 oocytes reached metaphase II, but this difference was not significant. Metaphase II oocytes in P1 were 34.3% more likely than those in TC199 to have a bipolar spindle with aligned chromosomes. Compared with oocytes at the germinal vesicle stage at 0 hour, those at metaphase I at 0 hour were more likely to progress to metaphase II (72.6% vs. 46.1% at 24 hours; 84.1% vs. 60.6% at 48 hours).

Conclusion(s): P1 is superior to TC199 for in vitro maturation of granulosa-free human oocytes.     

Live birth following vitrification of in vitro matured oocytes derived from sibling smaller follicles at follicle selection phase in the context of in vitro fertilization

In ovarian stimulation, a 31-year-old woman with polycystic ovary syndrome was at the risk of developing ovarian hyperstimulation syndrome, follicle aspiration was performed, and eight immature oocytes were collected from follicle fluids. After 28?h in vitro culture, six of them reached MII and were vitrified. The patient failed to conceive in her fresh in vitro fertilization cycle and next two replacement cycles. In the third replacement cycle, a successful pregnancy was obtained by vitrified-thawed oocytes. This case demonstrates that follicular aspiration during follicle selection phase has protective effects against developing ovarian hyperstimulation syndrome, and rescued immature oocytes are viable and could produce promising embryos for live birth.