The impact of vitrification on murine germinal vesicle oocyte In vitro maturation and aurora kinase A protein expression

Purpose

Investigate the effect of vitrification on in vitro maturation (IVM) and expression of Aurora kinases A, B, and C in germinal vesicle (GV)-stage oocytes.

Methods

GV-stage oocytes from B6D2F1 female mice 7–11 weeks of age were vitrified after collection, thawed, and matured in vitro for 0, 4, 8, and 12 h (hrs). The rate of germinal vesicle breakdown (GVBD), spindle apparatus assembly, and Aurora kinase mRNA and protein expression during IVM was measured.

Results

Oocyte vitrification was associated with significant delays in both GVBD and normal spindle apparatus assembly at 4 and 8 h of IVM (p < 0.05). There was no difference in mRNA levels between control and vitrified oocytes for any of the Aurora kinases. Aurora A protein levels were reduced in vitrified compared to control oocytes at 0 h (p = 0.008), and there was no difference at 4 and 8 h (p = 0.08 and 0.69, respectively) of IVM.

Conclusions

Vitrified oocytes have delayed GVBD and normal spindle assembly during in vitro maturation. Reduced levels of Aurora A protein immediately post-thaw may be associated with the impaired oocyte maturation manifested by the delayed progression through meiosis I and II, and the atypical timing of the formation of meiotic spindles in vitrified GV-stage oocytes.     

Quality evaluation of IVM embryo and imprinting genes of IVM babies

Purpose

Oxygen consumption rates of human embryos derived from in vitro matured (IVM) oocytes and controlled ovarian hyperstimulation (COH) were compared with scanning electrochemical microscopy (SECM) non-invasively in order to answer why embryos from IVM oocytes have lower developmental potential. We also analyzed the epigenetic disorders for IVM babies born in our clinic.

Methods

The oxygen consumption rate was calculated with the SECM system for different maturation stages of human oocytes, IVM and COH embryos. Blood from umbilical cords of IVM babies was collected to examine the imprinting genes.

Results

There were no significant differences in oxygen consumption of embryos at each cleavage stage between IVM and COH (range 0.26–0.56 × 10¹⁴/mol S⁻¹). There also was no abnormality found in expression of imprinting genes in IVM babies.

Conclusions

There are no differences in terms of oxygen consumption between embryos derived from IVM and COH. There was no imprinting gene disorder founded from IVM babies.     

In-vitro maturation of human oocytes: before or after vitrification?

Purpose

This study aims to determine if in-vitro maturation (IVM) of human immature oocytes should be performed before or after vitrification.

Methods

A total of 184 immature oocytes were randomly divided into two different groups: 100 were vitrified at metaphase II (MII) stage 24 h-48 h after IVM (group 1) and 84 were immediately vitrified at germinal vesicle (GV) or metaphase I (MI) stages and in vitro matured after warming (group 2).

Results

Survival rate after warming was similar in both groups (86.9% versus 84.5%). However, oocyte maturation rate per collected oocyte was significantly higher for oocytes matured before vitrification (group 1, 46%) than for oocytes vitrified before IVM (group 2, 23.8%) (p < 0.01). Consequently, the number of MII oocytes inseminated per oocyte collected was significantly higher for group 1 (40%) than for group 2 (23.8%) (p < 0.05).

Conclusion

IVM procedure is more efficient when it is performed before oocyte vitrification.     

Comparison of fertilization and embryonic development in sibling in vivo matured oocytes retrieved from different sizes follicles from in vitro maturation cycles

Purpose

To examine the fertilization and developmental potential of sibling mature oocytes collected from different follicle sizes on day of retrieval in in vitro maturation (IVM) cycles.

Methods

Two hundred thirty eight hCG-primed IVM cycles were performed in 213 patients with polycystic ovaries. If sibling mature oocytes were retrieved on day of collection, they were divided into two groups, Group 1 (n = 78): M-II oocytes obtained from follicles size 10–14 mm; Group 2 (n = 192): M-II oocytes obtained from follicles size <10 mm.

Results

Of the 238 cycles, 63 cycles had more than one M-II oocytes retrieved (total M-II oocytes = 270) both from Groups 1 and 2. There were no significant differences between the two groups for oocyte diameter (117.2 mm vs. 116.9 mm), fertilization (79.5% vs. 72.4%) and good quality embryo on day 3 (66.1% vs. 56.8%).

Conclusions

The M-II oocytes retrieved from the cohort of follicles (<10 mm diameter) can produce the same quality of embryos as that from large follicles, likely contributing to improve the clinical outcome.     

Spontaneous in vitro maturation of oocytes prior to ovarian tissue cryopreservation in natural cycles of oncologic patients

Purpose

To evaluate the recovery rate and spontaneous in vitro maturation (IVM) of immature oocytes enclosed within or released from follicles during the processing of ovarian tissue prior to its cryopreservation.

Methods

Thirty-three oncologic patients who had not previously undergone chemo or radiotherapy underwent ovarian tissue cryopreservation (OTC) during natural menstrual cycles. Immature oocytes, enclosed within follicles or released during ovarian cortex processing, were collected and matured spontaneously in vitro for 48 h. Nuclear maturation was assessed every 24 h and the ability of the IVM oocytes to display a normal activation response following parthenogenetic activation was evaluated. The following outcome measures were also evaluated: disease, age, FSH, LH, E2, P4 and AMH serum levels, menstrual cycle day, recovery and spontaneous IVM and parthenogenetic activation rates.

Results

Oocytes recovered per patient were 3.3 ± 0.7 (1.8–4.7 oocytes, 95CI), regardless of the menstrual phase. The mean number of IVM oocytes per patient was 1.3 ± 0.2 oocytes (95CI: 0.8–1.8), regardless of menstrual phase (p = 0.86) and oocyte origin (p = 0.61). Forty-one percent of oocytes extruded the second polar body and formed one pronucleus after parthenogenetic activation.

Conclusion

Twenty-one of the 33 women (63.6 %) requesting OTC produced at least one mature oocyte.     

In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising “ex vivo” method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe

Purpose

We present our center’s experience with 34 consecutive cases who underwent in vitro maturation (IVM) of oocytes obtained from ovariectomy specimens and compare our data with updated literature data.

Methods

Feasibility and efficiency of oocyte collection during ovarian tissue processing was assessed by the recovery rate, maturation rate, and embryological development after IVM.

Results

On average, 14 immature oocytes were retrieved per patient during ovarian tissue processing in 33/34 patients. The overall maturation rate after IVM was 36 %. The maturation rate correlated with the age of the patient and the duration of IVM. Predominately, oocyte vitrification was performed. Eight couples preferred embryo cryopreservation. Here, a 65 % fertilization rate was obtained and at least one good-quality day 3 embryo was cryopreserved in 7/8 couples. The retrieval of oocytes ex vivo resulted in mature oocytes or embryos available for vitrification in 79 % of patients. One patient with ovarian insufficiency following therapeutic embolization of the left uterine and the right ovarian artery because of an arteriovenous malformation had an embryo transfer of one good-quality warmed embryo generated after IVM ex vivo, which resulted in an ongoing clinical pregnancy.

Conclusions

IVM of oocytes obtained ex vivo during the processing of ovarian cortex prior to cryopreservation is a procedure with emerging promise for patients at risk for fertility loss, as illustrated by the reported pregnancy. However, more data are needed in order to estimate the overall success rate and safety of this novel approach.     

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development

Purpose

We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development.

Methods

Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates.

Results

No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0–3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21 % vs 48 %; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h.

Conclusions

This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.     

Fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle in vitro fertilization

Purpose

To analyze the fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle IVF in women with regular cycles.

Methods

Natural cycle IVF was performed in 28 patients who had normal ovaries, > 6 antral follicle counts and were less than 40 years old (n = 28 cycles). An hCG trigger of 10,000 IU was administered 36 h before oocyte collection when the diameter of the dominant follicle (DF) was over 12 mm. Oocytes were retrieved from DF as well as from the cohort of smaller follicles. Embryological aspects of the mature and immature oocytes retrieved from these cycles as well as the implantation and clinical pregnancy rates depending on the origin of the embryos transferred were evaluated.

Result(s)

Overall clinical pregnancy and implantation rates were 20.8 % and 6.7 %, respectively. There were no differences in in vitro maturation (IVM), fertilization and embryo development between immature oocytes retrieved with and without in vivo matured oocytes. However, the clinical and implantation rates in cycles with embryos produced from in vivo matured oocytes transferred were better than the cycles where only IVM embryos were transferred (30.8 %, 9.1 % vs. 9.1 %, 3.2 %).

Conclusion(s)

Although our results show that immature oocytes from natural cycle IVF can fertilize normally and can be used to increase the number of embryos available for transfer, the embryos derived from the immature oocytes in natural cycles IVF have a poorer reproductive potential.     

Supplementation with low concentrations of melatonin improves nuclear maturation of human oocytes in vitro

Purpose

Studies in bovine and porcine have indicated that melatonin (MT) could induce meiotic maturation of immature oocytes in vitro. The object of the current study was to investigate if MT could ameliorate human oocytes maturation during rescue in vitro maturation (IVM).

Methods

Two hundred seventy eight germinal vesicle (GV) oocytes and 451 (MI) metaphase I oocytes were vitrified, thawed and then matured in vitro. All the oocytes were randomly allocated into six groups in which the oocytes were cultured in medium supplemented with different concentrations of MT (0, 10⁻², 1, 10², 10⁴, 10⁶ nM) and nuclear maturation was evaluated at 6 h, 12 h, 18 h, 24 h and 48 h of culture.

Results

The optimal MT concentration for both GV and MI oocytes was 1 nM. At 24 h of culture, nuclear maturation rate of MI oocytes cultured in 1 nM MT medium was significantly higher than other groups (P < 0.05); Nuclear maturation rate of GV oocytes cultured in 1 nM MT medium was also significantly higher than the control group (P < 0.05). On the other hand, decreased nuclear maturation rate was observed in the high MT concentration group (10⁶ nM).

Conclusions

The current study demonstrated that low concentration of exogenous MT could ameliorate nuclear maturation of human oocyte during rescue IVM, while high concentration of MT presented negative effects.     

What is the optimal threshold of serum Anti-Müllerian hormone (AMH) necessary for IVM treatments?

Purpose

To assesse circulating levels of Anti-Müllerian hormone (AMH) as a predictor of oocyte number and their potential to mature in vitro in both normo-ovulatory (NO) women and in women with Polycystic Ovary Syndrome (PCOS) undergoing in vitro maturation (IVM) treatments.

Methods

We prospectively studied NO women and women diagnosed with PCOS, (age range 21–39 years) underwent IVM treatments at our center. Serum AMH levels were quantified before each cycle and correlated to oocytes number, maturation and fertilization during in vitro maturation.

Results

104 NO and 30 PCOS IVM cycles were followed with retrieval of a total of 672 and 491 oocytes, respectively. In NO women, the serum AMH level positively correlated with the number of oocytes retrieved, (R = 0.6; P <0.0001) the number of M2 oocytes at 24 and 48 h (R = 0.4; P <0.01; R = 0.26 p < 0.007, respectively) and with the total number of M2 oocytes (R = 0.47; P < 0.0001). In the PCOS group, the serum AMH level positively correlated only with the number of oocytes retrieved (R = 0.43; P <0.03). Receiver operating characteristic (ROC) analyses showed that a cutoff AMH level of 1.56 (ng/ml) could identify patients with 5 or more oocytes at OPU with a sensitivity of 83 % and a specificity of 75 %. An AMH level of 1.63 (ng/ml) was the threshold for 5 or more matured oocytes (sensitivity = 81 %, specificity = 53 %).

Conclusions

Serum AMH may be used as a marker to identify candidates for IVM treatment in both NO and PCOS women.     

The influence of amifostine administration prior to cyclophosphamide on in vitro maturation of mouse oocytes

Purpose

The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes.

Materials and methods

Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined.

Results

In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p < 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased.

Discussion

Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.     

Clinical outcomes from mature oocytes derived from preovulatory and antral follicles: reflections on follicle physiology and oocyte competence

Purpose

The aim of this retrospective study was to compare the competence of oocytes obtained from preovulatory and antral follicles.

Methods

Mature oocytes from preovulatory follicles were retrieved from women selected for standard IVF treatment (Group A). Mature oocytes from antral follicles were recovered from women undergoing hCG-primed in vitro maturation (IVM) treatment (Group B). Patients groups were matched for age, BMI, FSH, AMH and antral follicle count (AFC) values. In vivo matured oocytes from both groups were microinjected and resulting embryos were culture and selected on day 3 for embryo transfer.

Results

Oocyte pick-ups (OPU) were 315 and 204 in Groups A and B, respectively. Fertilization rates were comparable (72.8 and 75.9 %, respectively; P = 0.137). In Group A, in which the average number of embryos transferred was higher, clinical pregnancy rates per OPU (37.5 %) and embryo transfer (38.4 %) were superior in comparison to Group B (27.0 %, P = 0.013; 29.4 %, P = 0.041; respectively). On the other hand, implantation rates (Group A, 23.7 %; Group B, 20.8 %) and proportions of babies born per transferred embryo (Group A, 19.5 %; Group B, 16.9 %) were similar (P = 0.528 and 0.332, respectively).

Conclusions

Overall, this suggests that oocyte competence is already achieved at the antral stage of follicle development.     

Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining

Purpose

The present study was designed to investigate the effect of L-carnitine treatment during IVM on nuclear and cytoplasmic maturation of immature oocytes selected by Brilliant Cresyle Blue (BCB) staining, and their subsequent developmental competence.

Materials & methods

Compact cumulus-oocyte complexes (COCs) were collected from NMRI mice ovaries and stained with BCB staining. BCB+ (colored cytoplasm) oocytes were then cultured in tissue culture medium (TCM) 199 with 0.0, 0.3 and 0.6 mg/ml L-carnitine.

Results

The both L-carnitine concentrations significantly increased the intracellular glutathione (P < 0.001), nuclear maturation (P < 0.01) and expression levels of cyclin-dependent kinase1 (CDK1) (P < 0.05). Moreover, treated oocytes with 0.6 mg/ml L-carnitine showed increased (P < 0.05) expression of mitogen-activated protein kinase1 (MAPK1) mRNA. Also, adding L-carnitine (0.6 mg/ml) to IVM medium significantly increased the cleavage rate (P < 0.05). The blastocyst development rate (BDR) in the both L-carnitine treated groups was significantly higher (P < 0.001) than the control group. L-carnitine had no significant effect on total blastocyst cell numbers.

Conclusions

These data indicated that L-carnitine supplementation during IVM of immature BCB+ oocytes improved preimplantation developmental competence of oocytes after IVF, probably by accelerating cytoplasmic and nuclear maturation of oocytes. It may provide a novel approach to improving ART outcomes in infertile couples.     

Does IVF cleavage stage embryo quality affect pregnancy complications and neonatal outcomes in singleton gestations after double embryo transfers?

Purposes

Embryo quality is associated with successful implantation and live births. Our retrospective study was carried out to determine whether or not cleavage stage embryo quality affects the miscarriage rate, pregnancy complications and neonatal outcomes of singletons conceived with assisted reproduction technology.

Method

The current study included 11,721 In Vitro Fertilization-Embryo Transfer cycles (IVF-ET) between January 2009 (the date at which electronic medical records were implemented at our center) and March 2013. Only women < 40 years of age undergoing their first fresh embryo transfer cycle using non-donor oocytes were included.

Results

Our study indicated that the transfer of poor-quality embryos resulted in higher miscarriage (19.77 % vs. 13.28 %, p = 0.02) and lower ongoing pregnancy rates (15.33 % vs. 48.06 %, p < 0.001). Logistic regression analysis performed on data derived from 744 cycles culminating in miscarriages versus 4,333 cycles culminating in live births, suggested that embryo quality (p = 0.04) is significantly associated with miscarriage rate after adjusting for other confounding factors. Moreover, there were no differences in the mean birth weight, low birth weight (<2,500 g), very low birth weight (<1,500 g), gestational age, preterm delivery (<37 weeks), very preterm delivery (<32 weeks), congenital malformations, small-for-gestational-age singletons (SGA), and large-for-gestational-age singleton (LGA) rate (p > 0.05). Similarly, pregnancy complications resulting from poor-quality embryos were not different from good-quality embryos (4.04 % vs. 2.57 %, p = 0.33). Finally, logistic regression suggested that embryo quality was not significantly associated with pregnancy complications after adjusting for other confounding factors (p = 0.40).

Conclusions

Our study suggests that transfer of poor-quality embryos did not increase the risk of adverse outcomes; however, the quality of cleavage stage embryos significantly affected the miscarriage rate and ongoing pregnancies.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-014-0351-8) contains supplementary material, which is available to authorized users.     

Laparoscopic excision of ovarian endometrioma does not exert a qualitative effect on ovarian function: insights from in vitro fertilization and single embryo transfer cycles

Purpose

To evaluate whether laparoscopic excision of endometrioma exerts a qualitative effect on ovarian function.

Methods

A retrospective analysis of oocytes retrieved in 25 cycles of 21 patients undergoing IVF treatment with controlled ovarian stimulation. The number of oocytes recovered from ovaries with a history of excision of endometrioma (E-Ov) were compared to those from contra-lateral healthy ovaries (H-Ov) as for the analysis of a quantitative effect of surgery. As for the analysis of a qualitative effect, 55 oocytes from E-Ov were compared to 128 oocytes from H-Ov in terms of normal fertilization rate and the rate of top-quality embryos per normally fertilized eggs. Furthermore, 10 embryos derived from oocytes recovered from E-Ov were compared to 24 embryos derived from oocytes from H-Ov in terms of clinical and on-going pregnancy rates per embryos in 34 single embryo transfer cycles.

Results

Mean number of oocytes recovered from E-Ov was significantly smaller than that from H-Ov (2.2 ± 2.0 vs. 5.1 ± 3.3, P = 0.009). There was no difference between oocytes from E-Ov and H-Ov as for normal fertilization rate (63.6 % vs. 69.5 %, P = 0.43) and the rate of top-quality embryos (40.0 % vs. 49.0 %, P = 0.34). Clinical and on-going pregnancy rates per embryos were also similar in embryos derived from oocytes recovered from E-Ov and H-Ov (40.0 % vs. 25.0 %, P = 0.39 and 20.0 % vs. 20.8 %, P = 0.96).

Conclusions

The quality of oocytes recovered from the ovary with a history of laparoscopic excision of endometrioma is not inferior to the quality of oocytes from contra-lateral healthy ovary.     

Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption

Purpose

Only 50–60 % of immature human oocytes attain the mature stage in vitro. Such a deficiency may be a reflection of inadequate conditions of in vitro maturation (IVM) or a manifestation of intrinsic oocyte defects. In the present study, we explored the possibility that the DNA of immature oocytes may be damaged and that such a condition, or inability to trigger a repair action, is associated to germinal vesicle (GV) arrest.

Methods

Immature oocytes (GV-stage oocytes) were obtained from women undergoing stimulated (Stim-C) or IVM (IVM-C) cycles. GV oocytes obtained from stimulated cycles were fixed for successive analysis either after recovery (T0) or following 30 h (T30) of culture if still arrested at the GV stage. Oocytes retrieved in IVM cycles were used only if they were found arrested at the GV stage after 30 h (T30) of culture. All oocytes were fixed and stained to detect chromatin and actin. They were also assessed for positivity to γH2AX and Rad51, markers revealing the presence of double-strand DNA breaks and the activation of a DNA repair response, respectively. Labelled oocytes were analysed using a Leica TCS SP2 laser scanning confocal microscope.

Results

In Stim-C oocytes, γH2AX positivity was 47.5 and 81.5 % in the T0 and T30 groups, respectively (P = 0.003), while γH2AX-positive oocytes were 58.3 % in the IVM-C T30 group (Stim-C T0 vs. IVM-C T30, P = 0.178; Stim-C T30 vs. IVM-C T30, P = 0.035). Positivity for nuclear staining to Rad51 occurred in 42.1 and 74.1 % of Stim-C in the T0 and T30 subgroups, respectively (T = 0.006), while 66.7 % of IVM-C T30 oocytes resulted positive for a DNA repair response (Stim-C T0 vs. IVM-C T30, P = 0.010; Stim-C T30 vs. IVM-C T30, P = 0.345).

Conclusions

The present data document the existence of double-strand DNA breaks (DSBs) in human immature oocytes. Also, they are consistent with the hypothesis that insults to DNA integrity may be an important factor affecting meiotic resumption.     

Polar body fragmentation in IVM oocytes is associated with impaired fertilization and embryo development

Purpose

The significance of finding a fragmented first polar body in an oocyte prepared for ICSI is controversial with most recent publications suggesting that it is not prognostic for oocyte fertilization or embryo development. Our purpose was to look at this question in the context of oocytes not stimulated for conventional IVF.

Methods

Oocytes obtained for IVM and obtained from follicles at most 12 mm in diameter were evaluated for their polar body morphology soon after they entered metaphase II when they were denuded in preparation for ICSI. Records were evaluated retrospectively for the fertilization rate and the embryo growth rate (cell number) on each day of development for embryos with normal appearing polar bodies or fragmented polar bodies, but no other cytoplasmic dysmorphisms.

Results

Oocytes with fragmented polar bodies were significantly less likely to fertilize than oocytes with normal appearing polar bodies (p < 0.0001). Embryos which developed from oocytes with fragmented polar bodies had significantly impaired growth compared to embryos that developed from oocytes with normal appearing polar bodies (p = 0.0328).

Conclusions

Fragmented polar bodies likely reflect cytoplasmic incompetence.     

Follicular fluid Aβ40 concentrations may be associated with ongoing pregnancy following in vitro fertilization

Purpose

To determine whether Aβ40 levels in the follicular fluid (FF) of infertile women undergoing IVF demonstrate a relationship with IVF cycle parameters and outcome.

Methods

FF Aβ40 levels were compared between patients achieving ongoing pregnancy and those with unsuccessful cycles. Clinical data such as ongoing pregnancy rate, implantation rate, number of oocytes retrieved, number of 8 cells embryos with ≤5 % fragmants, ratio of 8 cells embryos with ≤5 % fragmants to total embryos per patient and cleavage rate were compared among three percentile groups of Aβ40. CCK-8 method was used to measure the effect of Aβ40 on rat granulosa cells proliferation in vitro. RT-PCR was used to detect the mRNA expression levels of steroidogenesis related genes.

Results

Patients achieving ongoing pregnancy (n = 26; 50.98 %) demonstrated significantly higher FF Aβ40 levels compared to those with unsuccessful cycles (n = 25; 49.02 %; P = 0.024). No significant differences were observed in APP (amyloid precursor protein) and its other proteolysis products including sAPPα, sAPPβand Aβ 42 between the two groups. Statistically significant differences between the three percentile groups of Aβ 40 were observed only in the implantation rates and ongoing pregnancy rates. There were no statistically significant differences between the three percentile groups in the age, No. oocytes retrieved, No. 2 pronucleus, No. embryos transferred, No. 8 cells embryos with ≤5 % fragmants and cleavage rate. Significantly negative correlation exists between APP and AFC (antral follicle count) (R =−0.360, P = 0.005) and oocytes retrieved (R =−0.378, P = 0.004). There were also significantly positive correlations between Aβ40 and Aβ42 (R = 0.407, P = 0.000), between AFC and oocytes retrieved (R = 0.476, P = 0.000). Rat granulosa cells treated with Aβ40 of different concentrations have improved their proliferative ability. Cells treated with 200 pg/ml Aβ40 have the strongest ability of proliferation. 200 pg/ml Aβ40 enhanced the expression of key molecules during steroidogenesis such as IGF-1,IGF-1receptor (IGF-1R),FSH receptor (FSHR),P450 aromatase (P450arom),steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochromes P450(P450scc).

Conclusions

Aβ40 levels in follicle fluid may be associated with ongoing pregnancy and the moderate expression level of Aβ40 is important for oocytes and embryos development.     

Follicular fluid protein content (FSH, LH, PG4, E2 and AMH) and polar body aneuploidy

Purpose

Our objective was to identify a marker for oocyte aneuploidy in follicular fluid (FF) in women with an increased risk of oocyte aneuploidy after controlled ovarian hyperstimulation.

Materials and methods

Three groups of oocytes were constituted for polar body screening by FISH (chromosomes 13, 16, 18, 21 and 22): Group 1, advanced maternal age (n = 156); Group 2, implantation failure (i.e. no pregnancy after the transfer of more than 10 embryos; n = 101) and Group 3, implantation failure and advanced maternal age (n = 56). FSH and other proteins were assayed in the corresponding FF samples.

Results

Of the 313 oocytes assessed, 35.78 % were abnormal. We found a significant difference between the follicular FSH levels in normal oocytes and abnormal oocytes (4.85 ± 1.75 IU/L vs. 5.41 ± 2.47 IU/L, respectively; p = 0.021). We found that the greater the number of chromosomal abnormalities per oocyte (between 0 and 3), the higher the follicular FSH level.

Conclusion

High FF FSH levels were associated with oocyte aneuploidy in women having undergone controlled ovarian hyperstimulation.