In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.

BACKGROUND: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential. METHODS: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 µg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section. RESULTS: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age. CONCLUSIONS: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries.

Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.

The oocyte population is not renewed in transplanted or irradiated adult ovaries.

BACKGROUND: According to conventional theory, the oocyte population is not renewed in mammalian ovaries after birth. A new hypothesis proposes that oocytes are generated continuously from haematopoietic progenitor cells. There is, however, no evidence that they can ovulate, although they may partially restore fertility by organizing 'helper follicles'. The hypothesis that follicles can form de novo in adult ovaries has been tested in a transplant model. METHODS: Ovaries from adult mice were transplanted under the kidney capsule or into the ovarian bursa of histocompatible, transgenic CAG::H2B-EGFP host animals. Some donors were sterilized before transplantation by X-irradiation to ensure 'empty niches' were available for repopulation. The phenotype of follicular oocytes at 2, 4 and 8 weeks post-transplantation was scored by epifluorescence. RESULTS: A total of 819 oocytes were examined in 30 ovarian grafts. None expressed green fluorescence, as would be predicted if they had formed de novo from germ cell progenitors in the systemic circulation of the host. Furthermore, small follicles eliminated by irradiation were not replaced in transplanted ovaries, and the few growing follicles present were apparently survivors of the original population. CONCLUSIONS: No evidence was found to support the hypothesis that progenitor cells from extra-ovarian sources can repopulate the adult ovary. The findings are consistent with the conventional view that a limited number of oocytes are formed before birth and declines with age. The study did not, however, rule out the possibility that germline stem cells may reside in the adult ovary.

A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.

BACKGROUND: A series of monozygotic (MZ) twin pairs discordant for premature ovarian failure presented an unusual opportunity to study ovarian transplantation. METHODS: Ten MZ twin pairs requested ovarian transplantation and eight have undergone transplantation with cryopreservation of spare tissue. Seven had a fresh cortical tissue transplant, one of whom received a second frozen-thawed transplant after the first ceased functioning at three years. One had a fresh microvascular transplant. RESULTS: All recipients reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 77-142 days. Six have already conceived naturally (one twice). Currently, two healthy babies have been delivered, and another three pregnancies are ongoing. The oldest transplant functioned for 36 months, resulting in one child and one miscarriage. She conceived again after a frozen-thawed secondary transplant. There was no apparent difference in return of ovarian function between the eight fresh ovarian grafts and the one frozen graft. CONCLUSIONS: Ovarian transplantation appears to restore ovulatory function robustly. Successful pregnancies, including one after cryopreservation, bode well for application to fertility preservation.

Luteinizing hormone requirements for ovulation in the pentobarbital-treated proestrous rat.

The LH requirements for ovulation in the pentobarbital-blocked proestrous CD rat have been studied by increasing serum gonadotropin levels through electrical stimulation of the brain and subsequently comparing the effects of timed hypophysectomy on ovulation and serum LH concentrations. The arcuate nucleus (ARC) or the medial preoptic area (POA) was stimulated unilaterally for 45 min with matched pairs of biphasic rectangular pulses through a coaxial platinum electrode. Serum LH was significantly elevated above basal values at the end of stimulation, but not in sham-stimulated controls. The results of both hormone measurement and hypophysectomy showed that the pituitary continued to release LH after extrinsic stimulation of the hypothalamus had ceased. Animals did not ovulate if they had been hypophysectomized at the end of the 45 min stimulation whereas nearly all ovulated if hypophysectomy was delayed for an additional 20 min. Some evidence suggested that the pituitary could be removed earlier without affecting ovulation if the rate of LH release was increased. The minimum peak LH concentration measured in rats that subsequently ovulated fully was 187 ng/ml, substantially lower than concentrations ordinarily attained in the spontaneous proestrous surge. When serum LH was insufficiently high to cause follicle rupture, there was nevertheless the resumption of meiosis and luteinization of the large ovarian follicles. Attempts were made to restore ovulability in animals presumed to have released a subovulatory quota of gonadotropin. Ovulation was obtained when such animals, prepared by hypophysectomy after the 45 min stimulation, had been bilaterally nephrectomized prior to stimulation. However, multiple injections of progesterone after hypophysectomy were without effect. The results are discussed in relation to variables that affect minimum requirements of LH for ovulation.

Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C.

We have previously demonstrated that ovarian function and fertility can be preserved in sheep after castration by autotransplantation of cryopreserved strips of ovarian cortex. In the current experiments we have investigated the long term survival of such grafts by detailed measurements of ovarian function for a period of nearly 2 yr after autotransplantation. After ovariectomy and transplantation of frozen/thawed grafts, the concentrations of FSH and LH rose to castrate levels for about 14 weeks before falling gradually to reach near-normal levels at about 60 weeks. In the breeding season from October 1994 to March 1995, all ewes had 5-10 estrous cycles that were similar in length to those in the 4 control ewes. Luteal function as indicated by the progesterone concentration was identical before and 11 months after transplantation. In contrast, the basal concentrations of FSH and LH were persistently raised throughout the luteal phase, but showed a normal decline during the follicular phase. The concentration of inhibin A in ovarian venous plasma measured at the end of the experiment 22 months after transplantation was significantly lower than that in control ewes (mean +/- SE, 409 +/- 118 vs. 1914 +/- 555 pg/ml; P < 0.004). Transplantation of frozen/thawed ovarian tissue to SCID mice demonstrated that about 28% of primordial follicles survived the procedure. All of the ovaries transplanted into sheep contained large antral follicles and/or cysts, but very few primordial oocytes when recovered at autopsy after 22 months. These results demonstrate that despite a drastic reduction in the total number of primordial follicles, cyclical ovarian function is preserved in sheep after autotransplantation of frozen/thawed ovarian tissue and provide experimental confirmation that such a technique could provide a means of preserving fertility in women undergoing chemo- or radiotherapy for malignant disease.

Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles.

FSH stimulates antral follicles to grow, but its role in earlier stages, if any, is obscure. Our aim was to determine the follicle stage at which the FSH receptor (FSHr) gene is first expressed. We used a PCR-based strategy to analyze single follicles ranging from primordial to multilaminar stages after isolation from human ovaries. Ovarian tissue was obtained from 11 women (age range, 25-33 yr) undergoing elective cesarean section. Tissue was partially disaggregated in medium containing 1% collagenase, and follicles were manually dissected free of stroma. Follicle stages were confirmed microscopically as primordial (nongrowing), primary (1 layer of cuboidal granulosa cells), or having 2 or more layers of granulosa cells. Rectus muscle and stromal tissue were used as negative controls. Messenger ribonucleic acid (mRNA) from each follicle was reverse transcribed, and the resulting cDNA was amplified by nested PCR using primers for FSHr and actin. None of the 9 primordial follicles expressed FSHr mRNA. Thirty-three percent of the primary and 2-layer follicles were positive for FSHr mRNA (4 of 12 and 3 of 9, respectively), as were 100% (n = 4) of the multilaminar follicles. The difference in FSH expression between the growing and primordial follicles was significant. This study shows for the first time that transcription of the FSHr gene begins at the earliest stages of follicular growth and indicates that FSH may have a hitherto unsuspected physiological role in preantral follicle development. In addition, this study demonstrates the practical feasibility of investigating the expression of other genes during human folliculogenesis.

Uptake and metabolism in vivo of tritiated oestradiol-17beta in tissues of ageing female mice.

The uptake in vivo of tritiated oestradiol-17beta has been compared in young and aged ovariectomized CBA/H-T6 mice by examining the levels of radioactivity in tissues 1 h after injection. The specificity of oestradiol uptake was demonstrated by previous treatment of some animals with either diethylstilboestrol or progesterone. The levels of radioactivity in whole tissue extracts were similar in both age groups for the uterus, hypothalamus, cerebrum and serum, but the uptake in the pituitary gland was significantly lower in old mice. There was no evidence of altered hormone metabolism with age.

Effect of inhibin immunoneutralization on steroidogenesis in rat ovarian follicles in vitro.

Inhibin is a putative gonadal paracrine factor produced by FSH-stimulated granulosa cells. To assess the paracrine function of inhibin further, preantral follicles (approx. 240 microns) with attached thecal cells were isolated mechanically from immature rat ovaries and cultured individually in vitro for 5 days in medium containing homologous serum and FSH. After 5 days, follicles had grown to preovulatory size (approx. 470 microns) with a commensurate increase in oestradiol secretion but not progesterone. Immunoneutralization of endogenous inhibin resulted in a significant decrease in oestradiol secretion and an increase in progesterone accumulation. When antiserum-treated follicles were supplemented with exogenous inhibin, oestradiol secretion was restored and progesterone accumulation was reduced. Aromatase substrate (androstenedione) levels were too low to measure, regardless of antiserum treatment. However, follicles treated with inhibin antiserum in the presence of exogenous androstenedione also exhibited oestradiol levels similar to untreated controls while progesterone accumulation remained elevated. We interpret these data as evidence that inhibin secreted by FSH-stimulated granulosa cells exerts a physiologically significant paracrine function in the follicle wall. Based on previous observations that inhibin stimulates androgen synthesis by isolated thecal/interstitial cells, it is proposed that granulosa-derived inhibin promotes thecal androgen synthesis and hence oestrogen synthesis during preovulatory follicle development. The antiserum-induced increase in progesterone accumulation is most probably explained by reduced metabolism of C21 steroid substrate to androgen in thecal/interstitial cells deprived of inhibin. It is concluded that inhibin is a physiological modulator of follicular steroidogenesis which exerts its effect at the level of thecal cell androgen synthesis.

Effects of inhibiting prolactin secretion on the maintenance of embryonic diapause in the suckling rat.

Treatment of rats with bromocriptine between days 5 and 8 after the post-partum mating resulted in suppression of serum prolactin levels and caused luteal regression. Although this treatment led to embryonic resorption when suckling was prevented by removing litters soon after birth, the diapausing embryos of animals nursing a litter of eight pups were unaffected by the treatment. These results suggest that the high levels of prolactin and progesterone in the circulation during lactation are not responsible for maintenance of the diapausing state.

Interruption of parturition in rats by morphine: a result of inhibition of oxytocin secretion.

Oxytocin secretion is inhibited by opioids, and oxytocin is important in parturition. The effects on parturition of morphine, a relatively selective mu-opioid receptor agonist, were studied in the rat. Morphine or vehicle with or without the opiate antagonist naloxone were administered immediately after the birth of the second pup and the subsequent course of parturition was recorded in a total of 80 rats. Both s.c. morphine (10 mg/kg) and intracerebroventricular (i.c.v.) morphine (18 micrograms through a previously implanted cannula) interrupted parturition, delaying the birth of the sixth pup after treatment to 187.3 +/- 35.9 (S.E.M.) min and 195.4 +/- 19.5 min respectively, compared with 46.4 +/- 3.7 and 66.1 +/- 17.5 min after vehicle alone. The dose of morphine given i.c.v. had no effect when given s.c. Naloxone given concurrently prevented the effects of morphine. Eventually the rate of parturition in the morphine-treated groups recovered. Perinatal pup mortality rate was not increased when morphine was given to the mothers, but it did inhibit the expression of normal intrapartum maternal behaviour. Pup mortality was increased 48 h post partum by morphine given during parturition, and it reduced the proportion of rats with normal maternal behaviour 24 h post partum. Morphine did not affect spontaneous or oxytocin-stimulated contractile activity of the parturient uterus in vitro. The concentration of oxytocin in trunk blood plasma was decreased 40 min after i.c.v. morphine (24.3 +/- 3.9 vs 39.3 +/- 6.5 pmol/l in controls), as was vasopressin (7.2 +/- 1.5 vs 19.7 +/- 4.5 pmol/l in controls). Intravenous infusion of oxytocin (2-5 mU/min for 144.3 +/- 8.2 min; total infused 448.5 +/- 61.9 mU) after i.c.v. morphine re-started parturition; all pups were born to these rats (mean time to pup 6, 110.3 +/- 12.7 min) before the i.v. vehicle-infused rats given i.c.v. morphine re-started (mean time to pup 6, 406.3 +/- 125.2 min). It is concluded that morphine given during parturition acts centrally through opioid receptors to inhibit oxytocin secretion, and impairs the expression of maternal behaviour. Reversal of the effects of morphine on parturition by i.v. oxytocin demonstrates the important role of oxytocin in fetus ejection and expulsion.

Ovarian aging, follicular depletion, and steroidogenesis.

The follicular population. Menopause occurs as a consequence of the continuous utilization of a fixed store of primordial follicles leading to almost total depletion at mid-life or sometimes earlier. The great majority of follicles that disappear are lost by atresia rather than by ovulation, and the rate of loss accelerates in the last decade of menstrual life. The numbers of growing follicles at a given age are correlated with those of the primordial stages, but there are always more being recruited than required for a single ovulation each month. The extent to which a dwindling number is responsible for the character of cycles of the menopausal transition remains unclear. Ovarian secretion. While menstrual cycles remain regular, circulating concentrations of estradiol and progesterone are relatively independent of age. On the other hand, serum levels of inhibin are substantially lower in women approaching menopausal age, probably reflecting smaller numbers of growing follicles at the beginning of the cycle. Alleviation of negative feedback on the pituitary gland results in a greater output of follicle-stimulating hormone (FSH), but the effects of chronic superstimulation on the aging ovary are not known. Follicular aging. Aging of long-lived oocytes could affect the developmental potential of the follicle unit as well as compromising the chances of late pregnancy. Another important field of investigation is therefore to determine the balance of responsibility between cumulated damage to molecules by toxins, on the one hand, and the effects of physiological aging and such epiphenomena as the changing hormonal or paracrine environments, on the other.

Oogenesis as a foundation for embryogenesis.

The majority of oocytes in postnatal ovaries are small, non-growing and reside in primordial follicles. They have to undergo a prolonged phase of growth and differentiation before nuclear and cytoplasmic maturation enables them to resume meiosis and undergo fertilization. A better knowledge of this phase of oogenesis is essential for understanding causes of oocyte pathology and optimizing methods for growing oocytes in vitro and for cryopreservation. There could also be spin-off discoveries for contraceptive strategies and pharmacologically controlling oocyte maturation. During oocyte growth, a molecular programme for development is assembled for the timely expression of mRNAs, some of which are expressed throughout oogenesis while others are 'masked' until or after meiotic maturation. Masking and stability in storage are largely due to a truncated poly(A) tail, controlled by regulatory sequences on the 3'-untranslated region (UTR) of the mRNA. Most maternal RNAs are degraded early in cleavage, there being a narrow overlap between persisting maternal mRNAs and activation of the embryonic genome. Accumulation of RNAs and proteins are not, however, the only major changes taking place during oogenesis. Cytoplasmic organelles multiply and redistribute, and there are epigenetic modifications of DNA for monoallelic expression of imprinted genes. The granulosa cells are obligatory for they provide physical support, nutrients and mediate the regulatory influences of gonadotrophic hormones. On the other hand, the oocyte actively influences the growth and differentiation of its granulosa cells. Thus, healthy embryos reflect the quality of both the oocyte and the granulosa cells.

Low temperature storage and grafting of human ovarian tissue.

A finite stockpile of germ cells forms in the human ovary before birth and is progressively utilised until it is almost exhausted at menopause in mid-life. Currently, there is no proven method for preventing wastage of this irreplaceable store, although cryopreservation provides an opportunity for long-term preservation of oocytes. This technology can potentially be used to conserve fertility in patients undergoing sterilising treatment or otherwise at risk of an early menopause. The structure of the ovary is well-suited to tissue storage because primordial follicles are abundant, developmentally dormant and located peripherally. Thin cortical slices of tissue can be prepared either from biopsies collected laparoscopically or by dissecting the cortex from the whole ovary. To test survival after freezing and thawing, tissues donated from women undergoing Caesarian section or gynaecological surgery were cooled slowly to liquid nitrogen temperatures in various cryoprotectant solutions and thawed rapidly. Three weeks after grafting under the renal capsule of immunodeficient SCID mice the majority of follicles were still viable. To test the procedure in human volunteers, small discs of ovarian tissue were autografted to the anterior uterus. After 3-4 months the tissues still contained follicles, including growing stages with PCNA-positive granulosa cells, but only about a quarter of the original follicle population had survived. In another study using either human xenografts or murine isografts, follicle survival rates were improved by administration of antioxidants to counteract ischaemia-reperfusion injury. Ovarian tissue banking should still be regarded as an experimental procedure, though recent results indicate that it has clinical potential.

In vitro growth of human primordial follicles from frozen-banked ovarian tissue.

The rarity of human oocytes frequently limits the success of assisted reproductive technology and delays research progress. Development of technologies to grow mature oocytes from the more abundant small follicles, perhaps after long-term storage at low temperatures, is a theoretically attractive solution to both problems. The length of the follicular growth span from the primordial to Graafian stage and changes in the trophic requirements of the cells, cellular interactions, morphogenesis and the sheer increase in bulk as the antrum forms are major challenges for cell culture technology. Even so, much progress has been made with animal follicles, and has begun with human tissue. A multi-step procedure, which reflects these changes, is perhaps the most likely to succeed. At present, the best strategy appears to be to initiate follicle growth in situ and isolate the follicles or granulosa-oocyte complexes once they have progressed to preantral stages. The final step is to mature the oocytes within their cumulus cells. The prospects of succeeding at each stage, and producing a fertile gamete at the end, are likely to be greater by preserving cellular interactions and the phenotype of follicle cells as these provide the physiological environment in which oocytes develop.

Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice.

OBJECTIVE: To determine the long-term survival of frozen-thawed human ovarian tissue as xenografts in severe-combined-immunodeficiency (SCID) mice. DESIGN: Animal study. SETTING: Animal and laboratory facilities at an academic center. PATIENT(S): Ovarian tissue obtained from a 27-year-old woman. INTERVENTION(S): Grafting of frozen-thawed ovarian tissue in SCID mice for 22 weeks. MAIN OUTCOME MEASURE(S): Follicle counts and growth by morphology and PCNA staining in frozen-thawed grafts and fresh controls. RESULT(S): All three grafts were recovered intact after 22 weeks. Their stroma was devoid of necrotic cells and contained healthy follicles. The ratio of primordial-total follicles decreased significantly after grafting (0.94 +/- 0.02 to 0.87 +/- 0. 01, control vs. grafting). Compared with controls, after 22 weeks of grafting, a higher percentage of follicles had initiated growth (5.6 +/- 2.4 vs. 12.5 +/- 1.9), but there was still a significant number of primordial follicles/graft (75 +/- 6.8). Follicle stages were similar between two groups; only primordial and one-layer follicles were seen in the xenografts. In the controls, except for one two-layer follicle, the most advanced follicle was at the one-layer stage. CONCLUSION(S): Human primordial follicles survive freeze-thaw and long-term xenografting procedures and retain their capacity to initiate growth. These findings encourage future attempts for human autologous ovarian transplantation.

Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?

OBJECTIVE: To review the potential for cryopreserving immature follicles either in situ or after isolation from ovarian stroma and to consider the options for fertility by transplantation or in vitro follicle growth. DESIGN: The problems of storing embryos and mature (metaphase II) oocytes were considered in light of the needs of patients to protect fertility before undergoing potentially sterilizing therapy for cancer. The evidence from the experimental biology literature showing that immature oocytes (prophase I) in primordial follicles can be cryopreserved successfully and transplanted to produce fertile eggs was reviewed. The review, which was compiled from MEDLINE and other bibliographic databases, is intended to emphasize the practical opportunities for this technology and the need for future research rather than to be a comprehensive treatment of the subject. CONCLUSION(S): The disappointing results obtained with the cryopreservation of oocytes at metaphase II and ethical concerns about embryo storage are giving impetus to the banking of ovarian tissue for patients who require conservation of fertility. The numbers of needy patients are growing as long-term survivorship after high-dose chemotherapy and bone marrow transplantation rises. More speculatively, if ovarian tissue banking becomes a proven effective method, young, healthy women may request storage of ovarian biopsy samples to keep their reproductive options open in midlife when oocyte fertility is declining. Although the cryotechnology is not yet perfected, the major question now is how to use the tissue most effectively after thawing. For the present, ovarian tissue cryopreservation is still at the experimental stage, but it holds the promise of valuable applications.

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization.

OBJECTIVE: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body. DESIGN: Prospective case study. SETTING: Research laboratories, university hospital. PATIENT(S): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI. MAIN OUTCOME MEASURE(S): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro. RESULT(S): Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X -15 cht -19 cht -22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21. CONCLUSION(S): The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.