Ovary and ovulation: DNA fragmentation of oocytes in aged mice

To investigate whether female fertility decreases with age dueto poor oocyte quality, we examined the presence of DNA fragmentationin ovulated oocytes from young, mature and aged mice. Oocytesfrom three age groups of female mice (7–8, 20–24and 40–48 weeks) were retrieved from the ovlducts 15 hafter human chorionic gonadotrophin (HCG) injection. Oocytesfrom each mouse were incubated in a CO₂ Incubator for 0–60h in human tubal fluid (HTF). After incubation, each oocytewas stained with the terminal deoxynucleotidyl transferase-mediateddUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation(interpreted as apoptotic changes) was significantly higherfor oocytes from aged mice, and the fertilization rate was significantlylower, compared with oocytes from young and mature mice. Ourresults suggest that DNA fragmentation of oocytes might be oneof the reasons for poor oocyte quality and lower fertility inthe aged group.     

The occurrence of aneuploidy in human: lessons from the cytogenetic studies of human oocytes

During the last 4 decades, the cytogenetic investigation of human oocytes has never stopped to progress, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome nondisjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidies in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies, whereas most of initial studies have failed to confirm any relationship between maternal age and aneuploidy in human oocytes.     

Non-meiotic chromosome instability in human immature oocytes

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25–45 years of age) and 24 IVF oocyte donors (18–33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.     

Fertility after age 45: From natural conception to Assisted Reproductive Technology and beyond

The introduction of in vitro fertilization (IVF) in 1978 – an accomplishment awarded with the 2010 Nobel Prize in Physiology or Medicine – has revolutionized the lives of millions of couples previously unable to conceive. Though initially applied primarily to young women with blocked Fallopian tubes, over the past three decades IVF has been increasingly used to combat age-related infertility. It has become clear that the decline in fertility with increasing female age is largely due to a rapid decrease in oocyte (egg) quantity and quality, with a higher proportion of oocytes displaying genetic abnormalities. Despite significant improvements in medical stimulation and laboratory culturing of embryos, IVF success once women reach the age of 45 is exceedingly rare. It has been well established through elegant clinical studies that, unlike the ovary, the reproductive capacity of the uterus does not diminish with age, even into the late 40s and 50s. With the use of oocytes from younger donors, women who are menopausal can attain very high rates of conception. The increased obstetrical risks in this population, which has a higher risk of underlying medical co-morbidities, must be considered prior to attempts at assisted conception, often in consultation with a multidisciplinary team of physicians. Finally, attempts to reverse the age-related decline in oocyte quality through micro-manipulation of the nucleus and cytoplasm have yielded disappointing results and are fraught with ethical concerns.     

Fidgetin knockdown and knockout influences female reproduction distinctly in mice

Microtubule-severing proteins(MTSPs),are a family of proteins which use adenosine triphosphate to sever microtubules.MTSPs have been shown to play an important role in multiple microtubule-involved cellular processes.One member of this family,fidgetin(FIGN),is also involved in male fertility;however,no studies have explored its roles in female fertility.In this study,we found mouse fidgetin is rich within oocyte zona pellucida(ZP) and is the only MTSP member to do so.Fidgetin also appears to int...     

The paradox of declining fertility but increasing twinning rates with advancing maternal age

BACKGROUND: Advancing female age is associated with declining fertility potential due to decreasing numbers and quality of oocytes but also with a distinct increase in dizygotic twinning rates, a phenomenon that has never been explained. METHOD: An analysis of follicle development was made in 959 spontaneous ovulatory cycles of 507 women. RESULTS: Multiple ovarian follicular development (>1 follicle >14 mm) and, by implication, multiple rather than single ovulations occurred in 105 women whose mean age (36.1 versus 34.6 years) and mean basal FSH concentrations (10.3 versus 7.7 IU/l) were significantly greater than those with monofollicular development (P < 0.01). The prevalence of multifollicular development increased with age. CONCLUSIONS: Dizygotic twinning must be associated with the development of >1 large follicle, which we found to be a significantly more frequent occurrence in older women. It is hypothesized that the response of pituitary release of FSH to the decreased negative feedback induced by impending ovarian failure often 'overshoots', causing multiple follicular development. In the presence of two good-quality oocytes, a twin pregnancy may result.     

Preservation of fertility in women affected by cancer. An audit of the cryopreservation services provided for women at Human Assisted Reproduction Ireland

There has been a remarkable improvement in survival rates for adult and childhood cancers, due to progress in cancer treatments. However multiagent chemotherapy and radiotherapy are associated with gonadal failure. Whereas sperm banking is commonly performed, female gametes are not so amenable to cryopreservation. Options available to women for fertility preservation include, embryo and oocyte cryopreservation. Our unit established an oocyte/embryo cryopreservation service for women affected by cancer in October 2003. Ninteen women have been referred and five (26%) women have had oocytes cryopreserved. The mean age of women in the group cryopreserved was 34 years (range 21-41). Their mean day 3 FSH was 6.2 IU (range 5.5-6.8). The mean dose of rFSH used was 1870 units (range 1500-2600). The mean number of oocytes cryopreserved was 8 (range 3-16). Preservation of fertility is of major concern to women affected by cancer. This service offers them hope for their future fertility.     

Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection

In this study, we compared the fertilization rate and embryo quality after intracytoplasmic sperm injection (ICSI) as they relate to oocyte morphology. A total of 654 ICSI cycles yielding 5903 metaphase II oocytes were observed. The oocytes retrieved in these cycles were divided into (i) normal oocytes, (ii) oocytes with extracytoplasmic abnormalities (dark zona pellucida and large perivitelline space), (iii) oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, and refractile body), (iv) oocytes with shape abnormalities, and (v) oocytes with more than one abnormality (double and triple abnormalities). Intracytoplasmic vacuoles and aggregates of smooth endoplasmic reticulum were not recorded separately. The fertilization rate and quality of morphologically graded embryos did not differ between the groups. There were 77 cycles where all transferred embryos were derived from abnormal oocytes, and 164 cycles where all embryos were derived from normal oocytes. These cycles were studied further. The two groups were comparable regarding mean female age, duration of infertility, duration of ovarian stimulation, number of ampoules of gonadotrophin injected, and number of oocytes retrieved. Two clinical pregnancy rates (44.4 versus 42.1%) and implantation rates per embryo (10.3 versus 13.2%) were similar. In conclusion, in couples undergoing ICSI, abnormal oocyte morphology is not associated with a decreased fertilization rate or unfavourable embryo quality. Furthermore, embryos derived from abnormal oocytes yield similar clinical pregnancy and implantation rates when transferred compared with embryos derived from normal oocytes.      

体外受精后异常受精的影响因素分析

目的探讨体外受精后单原核和多原核孕卵生成的影响因素，为降低异常受精率探寻可行的方法。方法应用卡方检验分析927个体外受精周期（608个IVF周期和319个ICSI周期），共计9718个卵细胞资料，研究多原核和单原核孕卵生成率与体外受精方式，女方年龄，超促排卵方案，HCG日血清E2水平和获卵数的关系；结果（1）体外受精中单原核孕卵生成率与体外受精方式、女方年龄、获卵数、HCG日血清E2水平、超促排卵方案均无显著性关系；（2）常规IVF周期中多原核孕卵生成率显著高于ICSI组，但．6-的受精率显著低于后者；当获卵数〉15个和HCG日血清E2〉4000pg／ml时，体外受精周期中多原核孕卵生成率显著升高；（3）ICSI组，随着女方年龄的增高，多原核孕卵生成率显著增高。结论体外受精中异常受精生成的机理和影响因素不尽相同，针对不同不孕人群选择合适的体外受精方式有助于提高正常受精率，降低异常受精率。     

Differential distribution of aneuploidy in human gametes according to their sex.

The cytogenetic study of human gametes is a new and important source of information because most chromosomal abnormalities originate from meiotic disorders. The frequency and type of abnormalities were analysed in both spermatozoa and mature oocytes. A total of 13,975 human sperm chromosome complements and 1897 oocyte chromosome complements were analysed. In the present study, pooled cytogenetic data on human gametes have been examined to determine and compare the distribution of non-disjunctions in male and female gametes. Human spermatozoa are characterized by a significant excess of hypohaploidy and an equal distribution of aneuploidies among all chromosome groups, whereas mature oocytes display an equal ratio of hypohaploidy to hyperhaploidy and a high variability in the distribution of non-disjunction: in particular, there is a significant over-representation of aneuploidies in both D and G chromosome groups. This indicates that non-disjunction is not a random event in female meiosis and, consequently, that there are differences in the meiotic process between the sexes. Meiotic and environmental factors which could explain the non-random malsegregation of chromosomes in female meiosis are discussed. The role of maternal age as a cause of aneuploidy is questioned.     

Thiabendazole-induced cytogenetic abnormalities in mouse oocytes

Of the various classes of human genetic disorders, aneuploidy is the most prevalent. Besides its association with maternal age and its predominant origin during maternal meiosis I, little is known about the etiology of aneuploidy. Although various classes of chemicals have been shown to induce aneuploidy in experimental systems, there is no definitive evidence for the role of chemically induced aneuploidy and adverse human health effects, particularly germ cell effects. Thus, it is important to understand the potential of chemicals for inducing aneuploidy in germ cells. There are conflicting data in the literature about the ability of thiabendazole (TBZ) to induce aneuploidy; therefore, we investigated the potential of TBZ for inducing aneuploidy in oocytes. Superovulated ICR female mice were administered 0, 50, 100, or 150 mg/kg TBZ by intraperitoneal injection. The frequencies and percentages of hyperploid oocytes were 0/472 (0), 2/410 (0.5), 6/478 (1.3), and 3/427 (0.7) for control, 50, 100, and 150 mg/kg TBZ, respectively. The difference between controls and the 100 mg/kg dose was statistically significant. Also, the proportions of ovulatory mice and the number of oocytes collected per ovulatory female were reduced in the TBZ groups relative to controls. Based on these results, we conclude that TBZ induces a small, but significant increase in the frequency of aneuploid oocytes at toxic doses that also impair ovulation. Environ. Mol. Mutagen. 29:367–371, 1997. © 1997 Wiley-Liss, Inc.     

Donor age is paramount to success in oocyte donation.

Several reports suggest increasing age in oocyte donors decreases the chances of in-vitro fertilization (IVF) success, while others describe no effect. The published data concerning gravidity and parity are similarly conflicting. To further address these questions, we retrospectively studied 445 consecutive donor IVF cycles at two large university-based IVF practices. Donor cycles were analysed for the number of oocytes retrieved, gravidity, parity, and age of the donor, and pregnancy outcome in recipients. The previous gravidity and parity of the donor were not associated with successful pregnancy in recipients. The number of oocytes retrieved was positively correlated with pregnancy. However, after adjusting for donor age, neither prior fertility nor the number of oocytes retrieved were significant predictors. In contrast, the donor's age was highly associated with recipient success. We conclude that the age of the oocyte donor is a significant predictor of pregnancy success and should be a major factor in selecting prospective candidates. The gravidity and parity of the donor are insignificant predictors, as are the total number of oocytes retrieved at the time of oocyte harvest.     

Current status of the cryopreservation of human unfertilized oocytes

Cryopreservation facilitates the long-term storage of oocytes from patients in danger of losing ovarian function and allows greater flexibility in fertility services for other patients. If some of the oocytes collected following ovulation stimulation are stored prior to fertilization, this alleviates many of the ethical concerns associated with embryo preservation. Concerns that cryopreservation could lead to disruption of the spindle and chromosomes, thus leading to genetic abnormalities of the offspring produced, mean that this procedure is not permitted in some countries. The recent spate of human live births from thawed oocytes has prompted the granting of the first licence allowing the use of thawed oocytes in the UK. However, the success rate of this procedure is still low and further research is required to refine these techniques and to develop new ones.     

How to overcome male infertility after 40: Influence of paternal age on fertility

The recent trend toward delayed parenthood raises major safety concerns because of the adverse effects of aging on couple fertility. Studies have demonstrated that aging clearly affects female fertility, but can also affect male fertility. Although several theories have been proposed, the exact mechanisms responsible for the observed age-related decline in male fertility remain to be elucidated. It has been shown that advanced paternal age (PA) is associated with reduced semen volume as well as, reduced sperm count, motility and morphology. Recent studies have also reported that paternal aging is associated with a significant increase in the prevalence of both genomic and epigenomic sperm defects. In the context of natural and intrauterine insemination (IUI) conception, advanced paternal age has been associated with lower pregnancy rates and increased rates of spontaneous abortion (independent of maternal age). In IVF and oocyte donation programs, a significant decrease in late blastocyst development has been seen in those cycles using spermatozoa of men older than 55. However, no significant relationship between paternal age and IVF or ICSI pregnancy rates has been observed.     

Fluorescent study of chromatin and tubulin in apparently unfertilized human oocytes following ICSI

In this study we examined 138 oocytes which were meiotically mature and, on light microscopic examination, contained either no or one pronucleus following intracytoplasmic sperm injection (ICSI). Oocytes were fixed and simultaneously stained for chromatin (Hoechst 33258) and the spindle (alpha-tubulin antibody). In nine oocytes, no sperm nucleus was observed. The remaining oocytes were separated into two groups following staining; (i) oocytes which had remained at metaphase II after ICSI (n = 74); and (ii) oocytes in which resumption of meiosis was observed after ICSI (n = 55). In all oocytes in which sperm chromatin was absent no resumption of meiosis had occurred and therefore parthenogenetic activation by the process of ICSI seems to be a rare event. In 17 out of 74 (23%) oocytes which remained at metaphase II, staining identified premature chromosome condensation (PCC) of the sperm chromatin (G1-PCC). Sperm nuclear decondensation or further transformation of the sperm chromatin was observed in 56 out of 74 (76%) oocytes which remained at metaphase II after ICSI and in 46 out of 55 (84%) oocytes which had resumed meiosis, indicating that initiation of sperm decondensation is independent of the resumption of meiosis in the oocyte. In contrast, transition of the sperm nucleus beyond the decondensed stage only occurred in association with resumption of meiosis in the oocyte (no pronuclei in metaphase II oocytes). The presence of both male and female pronuclei in 53% of oocytes which had resumed meiosis indicates that changes in sperm chromatin beyond the initial decondensation stage are dependent on cytoplasmic conditions which also permit female pronuclear formation.      

Diethylhexyl phthalate exposure impairs follicular development and affects oocyte maturation in the mouse

Diethylhexyl phthalate (DEHP) is an estrogen‐like compound widely used as a commercial plasticizer and present in medical devices, tubing, food containers and packaging. It is considered an endocrine disruptor and studies on experimental animals showed that exposure to DEHP can alter the function of several organs including liver, kidneys, lungs and reproductive system, particularly the developing testes of prenatal and neonatal males. Exposure to DEHP has been proposed as a potential human health hazard. This study assessed the effects of DEHP on folliculogenesis and oocyte maturation using the mouse as the experimental model. Newborn female mice were hypodermically injected with DEHP at doses of 20 and 40 μg/kg per body weight following different exposure regimens during the weaning period. We found that DEHP altered both folliculogenesis and oocyte development. In particular, DEHP exposure significantly decreased the number of the primordial follicles at pubertal and adult age by possibly accelerating the rate of follicle recruitment dynamics, reduced and/or delayed the level of imprinted gene methylation in the oocytes and increased metaphase II spindle abnormalities in oocytes matured in vitro. Furthermore, the weight of pups and litter size of mothers exposed to DEHP were significantly lower than controls. Finally, the number of primordial follicles appeared significantly reduced also in the F1 offspring at the adult age. These results show that DEHP may have a number of adverse effects on oogenesis, especially when exposure occurs during early postnatal age, arising concerns about the exposure of human female infants and children to this compound. Environ. Mol. Mutagen. 54:354–361, 2013. © 2013 Wiley Periodicals, Inc.     

Tissue-engineered follicles produce live, fertile offspring

Oocytes grown in vitro are of low quality and yield few live births, thus limiting the ability to store or bank the ova of women wishing to preserve their fertility. We applied tissue engineering principles to the culture of immature mouse follicles by designing an alginate hydrogel matrix to maintain the oocyte's 3- dimensional (3D) architecture and cell-cell interactions in vitro. A 3D culture mimics the in vivo follicle environment, and hydrogel-encapsulated follicles develop mature oocytes within the capacity for fertilization similar to that of oocytes matured in vivo. Embryos derived from cultured oocytes fertilized in vitro and transferred to pseudopregnant female mice were viable, and both male and female offspring were fertile. Our results demonstrate that alginate hydrogel-based 3D in vitro culture of follicles permits normal growth and development of follicles and oocytes. This system creates new opportunities for discovery in follicle biology and establishes a core technology for human egg banks for preservation of fertility.     

Cumulus cells are required for the increased apoptotic potential in oocytes of aged mice

Recent studies with female ICR mice have suggested that oocyte DNA fragmentation is one reason for poor oocyte quality and lower fertility associated with ageing. Since it was not determined if this increased 'apoptotic' potential in aged oocytes is due to changes within the oocyte itself or within the microenvironment of cumulus cells (CC) surrounding the germ cell, we sought to clarify if CC were required to affect the rate of apoptosis in oocytes maintained in vitro. Intact cumulus-oocyte complexes (COC) were retrieved by superovulation of virgin female ICR mice at 7 weeks ('young') or 34-35 weeks ('aged') of age. One-half of the COC in each group were incubated at 37 degrees C in human tubal fluid medium under paraffin oil for 24 h. The other half of the COC in each group were denuded of CC and incubated under the same conditions (denuded oocytes; DO). Following incubation, COC were stripped of adherent CC by gentle pipetting. All DO were then fixed and checked by light microscopy for morphological changes characteristic of apoptosis. In young mice, the presence of CC had no significant effect on oocyte death rate (18 +/- 9% and 14 +/- 6% apoptotic oocytes in COC and DO, respectively; P > 0.05). However, in aged mice the percentage of CC-enclosed oocytes that underwent apoptosis was significantly greater as compared to the death rate in DO (48 +/- 3% versus 19 +/- 8% apoptotic oocytes, respectively; P < 0.05). This increased death potential was due to the presence of CC since the occurrence of apoptosis in DO of aged versus young mice was not significantly different (19 +/- 8% versus 14 +/- 6% apoptotic oocytes, respectively; P > 0.05). These results demonstrate that the age-dependent acceleration of apoptosis in oocytes maintained in vitro requires the CC.      

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

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

Diethylstilbestrol (DES)-induced cell cycle delay and meiotic spindle disruption in mouse oocytes during in-vitro maturation

Due to the growing amount of data related to the deleterious effects of the synthetic oestrogenic compound, diethylstilbestrol (DES), on the female reproductive system, we tested the potential effects of this compound on mouse oocytes. Controlled time- and dose-dependent in-vitro experiments were carried out on isolated cumulus-oocyte-complexes (COCs) to examine the meiotic spindle assembly and chromosome distribution. alpha-tubulin, chromosomes and F-actin were labelled and detected by confocal laser scanning microscope. COCs were exposed to varying doses of DES (5-30 micromol/l) from the germinal vesicle (GV) stage to the end of metaphase II (MII) when meiosis I and meiosis II is normally completed. Exposure to DES during meiosis I caused a dose-dependent inhibition of cell cycle progression. In comparison with controls, fewer oocytes reached metaphase I (MI) at low doses (5 micromol/l) of DES, while none of the oocytes reached MI in high doses (30 micromol/l). When COCs were exposed to high doses of DES during meiosis II, fragmentation of first meiotic spindle was detected, whereas lower doses caused loosening of the first and the second meiotic spindles. No microtubular abnormalities were detected either in GV-stage oocytes or in cumulus cells. The above data demonstrate that one mode of action of DES on mouse oocytes is a severe yet reversible deterioration of meiotic spindle microtubule organization during maturation.