The role of menstrual cycle phase and AMH levels in breast cancer patients whose ovarian tissue was cryopreserved for oncofertility treatment

Purpose

To determine the factors that affect oocyte extraction efficiency when using the “combined procedure”. In the present “combined procedure” ovarian tissue cryopreservation and oocyte extraction from an isolated ovary, later used in In Vitro Maturation (IVM), are performed concurrently.

Methods

Data were analyzed retrospectively and obtained from the clinical records of 27 young breast cancer patients referred for fertility preservation.

Results

The patients’ mean age was 33.7 (±3.8) years, mean serum anti-Müllerian hormone (AMH) concentration was 3.5 (±2.1) ng/ml, and mean number of extracted oocytes was 8.3 (±6.1). The phase of menstruation (follicular or luteal) did not affect either the number of oocytes extracted (P = 0.99) nor oocyte survival or maturation rates. Likewise, the number of oocytes that could be extracted was not affected by the type of laparoscopic procedure (multiple-port or single-incision laparoscopy; P = 0.94) or the molecular subtype of breast cancer (either Luminal A or B; P = 0.52). Analysis revealed that the number of extracted oocytes was well-correlated with the patient’s AMH serum level and age (coefficient of correlation: 0.60 and −0.48, respectively).

Conclusion

We conclude that the outcome of the “combined procedure” primarily depends upon the patient’s serum AMH level and age. Importantly, the “combined procedure” may be used during any phase of the menstrual cycle to preserve the fertility of breast cancer patients.     

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.     

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.     

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.     

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.     

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.     

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.     

Experiences in fertility preservation: lessons learned to ensure that fertility and reproductive autonomy remain options for cancer survivors

Purpose

Assess fertility preservation (FP) measures chosen by patients newly diagnosed with malignancy and their outcomes.

Methods

Reproductive-age patients referred for FP underwent counseling and elected cryopreservation vs. no treatment. Outcome measures included ovarian stimulation, FP choice, oocytes/zygotes retrieved/cryopreserved and pregnancy outcome.

Results

From 2005 to 2012, 136 patients were counseled with 124 electing treatment: 83 oocyte-only, 21 oocyte + zygote and 20 zygote-only cryopreservation. Age, partnership and financial status factored into FP choice. Treatment was completed in 12 ± 2 days with 14 ± 11 metaphase-II oocytes harvested and cryopreserved/cycle. Eight patients returned to attempt pregnancy; three succeeded.

Conclusions

Our data demonstrate that oocyte and/or zygote banking are feasible FP options for women with malignancy; given the choice, the majority elected oocyte cryopreservation, highlighting desire for reproductive autonomy. Continued growth and research, combined with interdisciplinary communication, will ensure that appropriate candidates are offered FP and the potential for future parenthood, an important quality-of-life marker for survivors.     

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.     

Fifteen year follow-up of embryos cryopreserved in cancer patients for fertility preservation

Purpose

Determine the outcome of embryo cryopreservation in female oncology patients

Methods

The outcomes of IVF/ICSI cycles in oncology patients over 15 years in a University Teaching Hospital.

Results

Forty-two oncology patients (mean 31.9 ± 3.9 years) underwent embryo cryopreservation treatment (n = 33 IVF, n = 6 ICSI). Controlled ovarian stimulation with GnRH antagonist protocol (n = 34; 81 %) yielded fewer oocytes than GnRH agonist protocol (n = 8; 19 %) (9.4 ± 6.3 vs. 15.3 ± 8.9; p = 0.04) respectively. There was no significant difference in mean (±SD) duration of ovarian stimulation (11.6 ± 2.6 vs.10.6 ± 2.7), median gonadotrophin dose (1950 vs. 1670 IU), median day 5–6 oestradiol level (1124 vs.1129 pmol/l) or embryo yield (6.2 ± 4.1 vs. 8.8 ± 4.3; p = 0.07) between GnRH antagonist and agonist treatment cycles respectively. Thirty-nine patients cryopreserved embryos and three had their cycle cancelled. During this study period, of those who cryopreserved embryos, 5 patients underwent 9 frozen-thaw cycles (13 %), resulting in 2 live births (1 twin, 1 singleton, live birth rate 22 %). Six patients died (15 %), 3 conceived naturally (8 %) and 2 couples separated (5 %). Fourteen patients discarded their embryos (36 %). Twenty-two patients’ (56 %) have embryos remaining in storage.

Conclusions

This study demonstrates that embryo cryopreservation in female oncology patients gives a satisfactory live birth rate. However, there are concerns regarding cost-effectiveness, resulting from high disposal/non-usage of embryos, and further studies are required.     

A review of 15 years of ovarian tissue bank activities

Purpose

To review 15 years of activities in ovarian tissue cryobanking from medical database files, including patient indications, histological evaluation and clinical characteristics.

Methods

Retrospective longitudinal analysis of data from an ovarian tissue bank in an academic hospital. Five hundred and eighty-two patients had their ovarian tissue cryobanked between April 1997 and January 2012. Analysis of cryobanking database: precryopreservation patient characteristics, indications and safety issues, laboratory files and postcryopreservation clinical data.

Results

Of the 582 patients who had their ovarian tissue cryopreserved, 106 patients donated for research purposes and 476 patients for fertility preservation and long-term cryopreservation. Clinical data analysis of the 476 patients revealed a mean age at the time of cryopreservation of 23 ± 8.5 years (range: 9 months – 39 years), with 96.2 % of subjects aged ≤35 years (n = 458). Among 391 cases of malignant disease, hematological malignancies (39.9 %, n = 156) and breast cancer (21.7 %, n = 85) were the two main indications. At histology, malignant cells were found in ovarian tissue from leukemia patients (n = 3) and non-Hodgkin’s lymphoma patients (n = 2). Eleven patients underwent autotransplantation, resulting in 5 live births and 1 ongoing pregnancy.

Conclusion

This is the largest and most comprehensive study to describe and analyze indications and clinical patient characteristics before and after ovarian tissue cryopreservation. The procedure is safe, easy and promising. The database concept is a useful tool in patient selection for autotransplantation.     

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.     

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.     

Serum anti-Mullerian hormone levels correlate with ovarian response in idiopathic hypogonadotropic hypogonadism

Purpose

The role of serum AMH levels in prediction of ovarian response in idiopathic hypogonadotropic hypogonadism (IHH) was evaluated.

Material method(s)

Twelve patients with IHH underwent controlled ovarian hyperstimulation (COH) for IVF were enrolled in this prospective study. Serum AMH levels were studied on the 2nd or 3rd day of an induced menstrual cycle by a preceding low-dose oral contraceptive pill treatment. A fixed dose (150–300 IU/day) of hMG was given in all COH cycles. Correlations between serum AMH levels, COH outcomes and embryological data were investigated.

Results

Mean serum AMH levels was 3.47 ± 2.15 ng/mL and mean serum peak estradiol was 2196 ± 1705 pg/mL. Mean number of follicles >14 mm, >17 mm on hCG day and MII oocytes were 4.14 ± 3.2, 4 ± 2.5 and 7.28 ± 3.5, respectively. Mean number of grade A embryos and transferred embryos were 3.28 ± 2.4 and 2.5 ± 0.7, respectively. The clinical pregnancy rate per patient was 41.6 % (5/12). Positive correlations were observed between serum AMH levels and MII oocytes (r = 0.84), grade A embryos (r = 0.85), serum peak estradiol levels (r = 0.87), and number of follicles >14 mm (r = 0.83) and >17 mm (r = 0.81) on hCG day, respectively.

Conclusion

AMH appears as a promising marker of ovarian response in patients with IHH undergoing IVF.     

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.     

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.     

Ovarian response to controlled ovarian hyperstimulation in women with cancer is as expected according to an age-specific nomogram

Purpose

To evaluate the ovarian response to controlled ovarian hyperstimulation (COH) in cancer patients according to an age-specific nomogram for the number of retrieved oocytes.

Methods

Retrospective observational study carried out in a University affiliated fertility clinic. Forty-eight patients with cancer underwent ovarian stimulation for oocyte cryopreservation. An age - specific nomogram for the number of retrieved oocytes was built with 1536 IVF cycles due to male factor exclusively, oocyte donation and age related fertility preservation. The number of oocytes retrieved in cancer patients was compared to the expected response according to the nomogram using the Z-score.

Results

The mean number of total retrieved oocytes in patients with cancer was 14.04 ± 8.83. After applying the Z-score to compare the number of retrieved oocytes between women with cancer and the expected response according to the age-specific nomogram, we did not observe a statistically significant difference (Z-score 0.23; 95 % CI [−0.13-0.60]).

Conclusion(s)

According to our results, patients with cancer exhibit an ovarian response as expected by age. Despite the limitation of the sample size, the obtained results should encourage oncologists for early referral of women with cancer to fertility specialists.     

The association between the oocyte pool and aneuploidy: a comparative study of the reproductive potential of young and aged mice

Purpose

The present study examined the effect of aging on female reproductive potential.

Methods

Six-week-old and 9-month-old CD1 mice were referred to as the ‘young’ and ‘aged’ groups, respetively. Oocytes were collected after superovulation, and their viability were compared using parthenogenetic activation. The aneuploidy of the oocytes (MII) was assessed using chromosome spread, and the whole ovarian follicle number was counted using an unbiased stereological method. Serum hormone levels were measured using the radio-immunity method, and the expression of the Cohesin subunit genes in the oocytes (GV) were assessed using RT-PCR.

Results

The mean number of recovered (25.8 vs. 16.2; P < 0.05) and live oocytes (24.0 vs. 11.73; P <0.01) per head in the young-mice group (6-week-old) was significantly higher than that of the aged group (9-month-old). The aneuploidy rate of the ovulated oocytes in the aged group was significantly higher than that of the young group (36.8 % vs. 10 %; P < 0.01), and the rate of blastocyst formation in the young group (85.23 %) was significantly higher than that of the aged group (81.2 %; P <0.05). The number of primordial follicles (the oocyte pool) per ovary in the aged group was significantly decreased compared with the young group (330 ± 33.51 vs. 2079.6 ± 420.70; P < 0.01), and the level of AMH in the aged group was significantly higher than that of the young group (4.66 ± 0.11 ng/ml vs. 4.07 ± 0.18 ng/ml; P < 0.01).

Conclusions

We propose that maternal aging significantly reduces the oocyte pool, superovulation efficiency and developmental potential and increases the oocyte aneuploidy rate.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-013-0160-5) contains supplementary material, which is available to authorized users.     

Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure

Purpose

To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex.

Methods

Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro.

Results

Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p > 0.05 versus fresh control). 70.5 ± 5.2 % of follicles retained an intact morphology after cryopreservation (p = 0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p < 0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17β-oestradiol significantly increased during in vitro culture.

Conclusions

This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.