Ten-year experience with preimplantation genetic diagnosis (PGD) at the New York University School of Medicine Fertility Center.

We describe our experience of over 300 cycles of preimplantation genetic diagnosis (PGD) and report clinical pregnancy rates (35%-67%) that support using this technology to screen for genetic disorders and chromosomal abnormalities. In clinical practice for over ten years, PGD offers couples the earliest form of genetic screening and may help improve ongoing pregnancy rates in poor-prognosis patients.

Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea.

Population genetic structures of the mackerel (Scomber scombrus) and chub mackerel (Scomber japonicus) were studied in the Mediterranean Sea. Fragments of 272 bp (S. scomber) and 387 bp (S. japonicus) of the 5'-end of the mitochondrial control region were sequenced from spawning individuals collected off the coasts of Greece, Italy, Spain, and Portugal. High levels of mitochondrial control region haplotypic diversity (> 0.98) were found for both Scomber species. Nucleotide diversity was higher in the mackerel (0.022) than in the chub mackerel (0.017). Global F(ST) values were also higher and significant in the mackerel (0.024, P < 0.0001) as opposed to the chub mackerel (0.003, P > 0.05). Molecular variance analyses showed differential genetic structuring for these two closely related species. There is extensive gene flow between Mediterranean Sea and Atlantic Ocean populations of chub mackerel, which are organized into a larger panmictic unit. In contrast, Mediterranean Sea populations of mackerel show some degree of genetic differentiation and are structured along an east-west axis. The analysed eastern Mediterranean Sea mackerel populations (Greece, Italy) are clearly separated from that of the western Mediterranean Sea (Barcelona), which forms a panmictic unit with eastern Atlantic Ocean populations. The genetic structures of both species showed asymmetric migration patterns and indicated population expansion.

Preimplantation genetic diagnosis (PGD), a collaborative activity of clinical genetic departments and IVF centres.

Preimplantation genetic diagnosis (PGD) requires the combined efforts of geneticists and workers in the field of reproductive medicine. This was studied on the basis of a questionnaire, sent to 35 members of the PGD Consortium of the European Society of Human Reproduction and Embryology (ESHRE). A reply was obtained from 20 centres. They represent the majority of activities in the field of PGD in the world. It is obvious that many of the activities (in vitro fertilisation, embryo culture and biopsy) take place in IVF units while others (counselling and diagnosis) are the responsibility of genetic diagnostic centres. The distances between both units vary considerably. In all but one centre sex determination is offered. Aneuploidy screening is offered in 13 out of 20 centres. PGD of translocations and other structural chromosome abnormalities is offered in all but one centre. The number of monogenic diseases offered varies considerably. In comparison to prenatal diagnosis PGD is more expensive. The majority of these costs are due to the IVF or ICSI procedure. The charges for PGD vary between about 600 euro and 4000 euro. In 16 out of 20 centres the parents to be must sign an informed consent form.

Ooplasmic influence on nuclear function during the metaphase II-interphase transition in mouse oocytes.

Nuclear and pronuclear transfer procedures were used to assess the functional competence of the nucleus and cytoplasm of mouse germinal vesicle-stage oocytes denuded of granulosa cells and matured in vitro or in vivo before artificial activation using a sequential treatment of A23187 + cycloheximide. Following activation, in vitro-matured oocytes were "fertilized" by inserting a male pronucleus (PN), cultured to the 2-cell stage, and then transferred to the oviducts of foster mothers. No live births were noted, whereas a 17% live birth rate was observed when in vivo-matured oocytes were used. The developmental competency of other zygotes was similarly assessed following the exchange of haploid PN of matured and activated eggs with the female PN of fertilized zygotes. When PN of oocytes subjected to maturation and activation in vitro were transferred, only 1 of 79 reconstructed zygotes developed to term. In contrast, the live birth rate was 21% (11 of 53) for zygotes reconstructed with PN from in vivo-matured oocytes. Moreover, a live birth rate of 23% (8 of 35) was observed for reconstructed zygotes with female PN from "hybrid" oocytes created by transferring the metaphase II nuclei of in vitro-matured oocytes into enucleated, in vivo-matured oocytes before activation. Such results suggest that the nucleus of an in vitro-matured oocyte can support embryonic development, but only when it is activated in the proper ooplasmic milieu. The cellular factors creating this ooplasmic milieu appear to develop normally in vivo during follicle maturation to metaphase II, but they fail to do so when the oocytes are denuded of granulosa cells and cultured in vitro before the final stages of maturation. In parallel studies, male and female PN of in vivo-fertilized zygotes were inserted into oocytes that were activated and enucleated following either in vitro or in vivo maturation. Live birth rates were comparable at 19% (5 of 27) and 18% (9 of 49), respectively, suggesting that, regardless of the environment of the final stages of oocyte maturation, the resultant ooplasm is competent to support all aspects of embryonic development once activation and PN formation has been completed. Such findings only point further toward the importance of the condition of the ooplasmic milieu at the time of chemical activation. Whether a similar situation exists when eggs are activated following sperm penetration remains to be determined.

Fertility and maternal age strategies to improve pregnancy outcome.

In humans, the live birth rate drops precipitously with increasing maternal age, and this decline is associated with increases in the incidence of oocyte and embryo aneuploidy. Preimplantation aneuploidy screening has improved pregnancy outcome by significantly lowering the miscarriage rate. Nevertheless, aneuploidy screening only identifies the affected embryos; it does not attempt to correct the underlying biologic problem. Anomalies in chromosome segregation can result from a dysfunctional first or second meiotic division in the egg or develop after fertilization during the first few mitoses of early embryonic development. In both instances, ooplasmic anomalies may account for the nuclear problem. Low cell levels of cytoplasmic proteins (e.g., cytoskeletal elements, enzymes, energy stores, cell cycle regulatory proteins) may lead to a dysfunctional division of chromosomes during egg maturation or following fertilization. Ooplasmic injection is a micromanipulation technique that has produced pregnancies in patients with a history of poor-quality, fragmented embryos. Germinal vesicle transfer is a research procedure used to investigate the ooplasmic-nuclear interplay regulating cell cycle, maturation, and fertilization. Both these techniques may prove to be effective in improving the quality of eggs from patients of advanced maternal age.

Factors useful in predicting the success of oocyte donation: a 3-year retrospective analysis.

OBJECTIVE: To establish prognostic relevance of parameters assessed in oocyte donation cycles. DESIGN: Retrospective analysis. SETTING: Large university-based donor oocyte program. PATIENT(S): All oocyte recipient cycles achieving embryo transfer from September 1995 to October 1998. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy. RESULT(S): Recipient age and reproductive status, day 9 and 12 serum estradiol (E(2)) levels and a progesterone (P) level obtained 2 days after initiation of hormonal therapy did not correlate with pregnancy. Endometrial thickness, but not endometrial pattern, was useful in predicting pregnancy outcome. The clinical pregnancy and live-birth rate in cycles where the endometrial thickness was less than 8 mm was significantly lower when compared to cycles with an endometrial thickness > or =9 mm. Cycles where optimal quality embryos were transferred had the highest implantation (36%), clinical pregnancy (63%) and live birth (54%) rates and these rates were significantly higher than those of cycles where only poor quality embryos were available for transfer (10% implantation, 17% clinical pregnancy, and 8% live birth rates, respectively; P<.05). CONCLUSION(S): The most reliable predictive factors for pregnancy in oocyte donation cycles are the quality of the embryos transferred and the recipient's mid-cycle endometrial thickness. Recipient monitoring should minimally include ultrasound assessment of endometrial thickness.

A two- versus three-embryo transfer: the oocyte donation model.

OBJECTIVE: To compare implantation and pregnancy rates in oocyte recipients undergoing a two-embryo versus three-embryo transfer, 3 days after retrieval. DESIGN: Retrospective comparative analysis. SETTING: University-based in vitro fertilization center. PATIENT(S): All oocyte recipients undergoing embryo transfer from January 1, 1997 through August 31, 1999. INTERVENTION(S): Recipients received two or three embryos. MAIN OUTCOME MEASURE(S): Implantation, and clinical and multiple pregnancy rates. RESULT(S): Seventy-three recipients underwent a two-embryo transfer, and 376 had three embryos replaced. The numbers of oocytes retrieved (12.7 +/- 0.89 vs. 13.1 +/- 0.36) and embryos obtained (8.05 +/- 0.65 vs. 8.77 +/- 0.27) did not differ between the two-embryo and three-embryo transfer groups, nor did the proportion of patients with embryo cryopreservation (54.3% vs. 42.6%, respectively). There was no significant difference in pregnancy or implantation rates when comparing those patients with a two-embryo transfer to those with a three-embryo transfer. Significantly, 13.8% of the pregnancies in the three-embryo transfer group were triplet. CONCLUSION(S): Reducing the number of embryos transferred in an oocyte donation cycle can lower the incidence of triplet pregnancies without significantly lowering the overall pregnancy rate.

Analysis of Oct-4 expression and ploidy in individual human blastomeres.

Oct-4, a decisive factor that maintains totipotency in murine embryonic and germ cells, is exclusively expressed in such cells. In mice, different levels of oct-4 expression in blastomeres predict development towards inner cell mass (ICM) (high oct-4) or trophectoderm (TE) (low oct-4). To address whether the mouse model also applies to human embryos, the cytoplasm of individual human blastomeres from normally and abnormally fertilized embryos was tested for Oct-4 expression by reverse transcription-polymerase chain reaction (RT-PCR). The nuclei of the same blastomeres were subjected to fluorescence in-situ hybridization (FISH) to determine ploidy. A significant difference in Oct-4 mRNA levels was revealed between blastomeres. The distribution of blastomeres with high Oct-4 levels varied according to the cleavage stage of the embryo: the more blastomeres, the lower the percentage with high Oct-4 levels. Aneuploid blastomeres did not exhibit lower Oct-4 mRNA levels than diploid ones. Thus, differential Oct-4 expression in individual human blastomeres appears to direct cells towards the ICM or TE lineages without regard to chromosomal status. Oct-4 might be used as a marker in preimplantation genetic diagnosis to identify embryogenic blastomeres.

Oct-4 expression in inner cell mass and trophectoderm of human blastocysts.

The expression of the transcription factor Oct-4 is thought to be one of the decisive factors that maintain totipotency in embryonic and germ cells. In mice, oct-4 is exclusively expressed in germ cells and totipotent cells of the embryo. In humans, Oct-4 is expressed in germ cells, embryonic stem cells and whole embryos at various stages of development. However, there is limited information about the distribution of Oct-4 expression in human embryos. In an attempt to address this issue, the inner cell mass (ICM) and trophectoderm (TE) of 17 human blastocysts were separated and Oct-4 mRNA expression individually assessed by reverse transcription-polymerase chain reaction (RT-PCR). In discarded blastocysts that developed from two pronuclear zygotes, the mean Oct-4 expression was 31 times higher in totipotent ICM cells than in differentiated TE cells. This finding suggests that, in accordance with data from the mouse, Oct-4 is highly expressed in human ICM cells as opposed to TE cells; this in turn supports the hypothesis that Oct-4 plays a similar role to maintain totipotency in these two species.

In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei.

We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa.

The Herlitz type of junctional epidermolysis bullosa (H-JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H-JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8-cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Chronic 'binge' pattern cocaine alters the neuroendocrine profile of pregnant rats.

Pregnant Fischer rats injected with cocaine in a 'binge' pattern (3x15 mg/kg, i.p.) from gestational days 8 through 17, had significantly higher levels of progesterone (212.12+/-22.50 vs. 91. 99+/-15.41 ng/ml) and corticosterone (257.99+/-21.76 ng/ml vs. 31. 70+/-7.93, respectively) than saline-treated dams. No significant differences in prolactin were observed (2.36+/-0.17 vs. 2.17+/-0.19 ng rPrl132/ml). Correlation analysis indicated that there is a significant relationship between plasma levels of progesterone and corticosterone and the quality of nests built by the dams. No correlation was found within animals between prolactin plasma levels and the nest quality. Thus, cocaine's effect on progesterone and corticosterone may contribute to the series of behavioral alterations associated with cocaine exposure during pregnancy.

The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1.

OBJECTIVE: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti-human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the motility and vitality of human spermatocytes. DESIGN: Prospective, controlled study. SETTING: New York University School of Medicine. PATIENT(S): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. INTERVENTION(S): Human sperm were treated with the anti-HIV agents, MAP30 and GAP3 1. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. MAIN OUTCOME MEASURE(S): The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. RESULT(S): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100-0.1 microg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. CONCLUSION(S): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases.

Electrical activation and in vitro development of human oocytes that fail to fertilize after intracytoplasmic sperm injection.

OBJECTIVE: To determine whether electrically stimulated Ca2+ influx can "rescue" fertilization and early embryogenesis in human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). DESIGN: Prospective, randomized trial of a laboratory procedure. SETTING: A research laboratory at a university medical center. PATIENT(S): Discarded oocytes from ICSI-IVF cycles. INTERVENTION(S): Oocytes (n = 104) that showed no evidence of fertilization 16-24 hours after ICSI were assigned to three treatment groups: group 1 (one direct current electrical pulse at 1.36-1.50 kV/cm for 40-60 micros), group 2 (three pulses every 15-20 minutes), or group 3 (treated the same as group 2 but with no electrical stimulation). MAIN OUTCOME MEASURE(S): After stimulation, the oocytes were cultured in vitro for 3-5 days. Oocytes that displayed two pronuclei and a second polar body within 16 hours were considered to have fertilized normally. Fertilization and embryo cleavage rates were compared between groups. RESULT(S): Fertilization occurred in 26 (70%) of 37 and 38 (78%) of 49 group 1 and 2 oocytes, respectively, but in only 5 (27%) of 18 group 3 oocytes. Within 3 days, group 2 embryos routinely developed beyond the two-cell to four-cell stage (61% versus 13% in group 1); 11% of these oocytes developed to the morula or early blastocyst stage. Sex chromosome analyses indicated 10 male and 8 female embryos. CONCLUSION(S): Oocytes that fail to fertilize by 24 hours after ICSI can resume apparently normal fertilization and early embryonic development in response to electrical stimulation. Moreover, the degree of cytoplasmic activation as determined by the number of pulses applied affects fertilization efficiency and early embryonic development.

Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

In vitro fertilization outcome relative to embryo transfer difficulty: a novel approach to the forbidding cervix.

OBJECTIVE: To assess the impact of ET difficulty on IVF outcome and to optimize the ET procedure. DESIGN: Retrospective analysis of IVF outcome by ET catheter type and ET difficulty. Prospective treatment and follow-up of patients with a history of extremely difficult cervical passage. SETTING: Large university-based IVF program. PATIENT(S): All patients < 40 years of age undergoing IVF-ET from September 1995 to May 1998. INTERVENTION(S): Surgical correction of cervical stenosis. MAIN OUTCOME MEASURE(S): Pregnancy and embryo implantation rates. RESULT(S): Only 0.6% of ETs were "extremely difficult." Pregnancy rates were not statistically significantly different among ETs graded easy, moderate, and difficult. In contrast, no pregnancies occurred in the rare "extremely difficult" ET group. Eight patients with a history of extremely difficult cervical passage underwent surgical correction of their cervical stenosis. Twelve postoperative IVF-ET in these women resulted in eight clinical pregnancies, six of which were multiple gestations. The embryo implantation rate of these cycles was 42.2%. CONCLUSION(S): Patients with a history of extremely difficult ET may benefit from hysteroscopic evaluation and possible modification of their cervical canal before a future IVF attempt.

Oral versus intramuscular progesterone for in vitro fertilization: a prospective randomized study.

OBJECTIVE: To evaluate the efficacy of oral micronized progesterone compared with IM progesterone in oil for luteal support in patients undergoing IVF who are treated with a GnRH agonist. DESIGN: Randomized prospective clinical trial. SETTING: University-based IVF center. PATIENT(S): Women <40 years of age who were undergoing IVF with luteal GnRH pituitary down-regulation. INTERVENTION(S): Patients were randomized to receive either oral micronized progesterone (200 mg three times daily) or IM progesterone (50 mg daily). MAIN OUTCOME MEASURE(S): Progesterone levels at standardized days 21 and 28, and pregnancy and embryo implantation rates. RESULT(S): Day 21 progesterone levels were 77.6+/-13.2 ng/mL in the IM group and 81.5+/-16.2 ng/mL in the oral group. Day 28 progesterone levels were 76.3+/-15.0 ng/mL in the IM group and 53.6+/-10.1 ng/mL in the oral group. The clinical pregnancy rates were 57.9% and 45.8% for the IM and oral groups, respectively. The implantation rate per embryo was significantly higher in the IM group (40.9%) than in the oral group (18.1%). CONCLUSION(S): When used according to our protocols, oral progesterone and IM progesterone result in comparable levels of circulating progesterone. However, oral progesterone results in a reduced implantation rate per embryo.

Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy.

OBJECTIVE: To determine whether baseline serum FSH and/or E2 concentrations can predict the risk for fetal chromosomal abnormalities. DESIGN: Case control study. SETTING: Reproductive technology program at a university hospital. PATIENT(S): Patients who underwent dilation and curettage (D + C), and whose products of conception were karyotyped. INTERVENTION(S): Patients underwent natural conception or controlled ovarian hyperstimulation followed by intrauterine insemination, in vitro fertilization and embryo transfer, gamete intrafallopian transfer, or zygote intrafallopian transfer. MAIN OUTCOME MEASURE(S): Baseline serum FSH and E2 concentrations and fetal karyotype. RESULT(S): Genetic evaluation of 78 D + C specimens revealed 34 normal and 44 abnormal fetal karyotypes. A significantly greater proportion of women with abnormal fetal karyotype had elevated baseline serum FSH (> or =15 mIU/mL [RIA] or 10 mIU/mL [Immulite]) and/or E2 > or = 50 pg/mL [Immulite]) compared with women of normal fetal karyotype. Among karyotypically abnormal abortuses, autosomal trisomy was the most common abnormality noted (79.5%), followed by mosaicism (6.8%), triploidy (6.8%), monosomy XO (4.5%), and balanced translocation (2.3%). CONCLUSION(S): Baseline serum FSH and/or E2 concentrations may be valuable as predictors of fetal aneuploidy.

In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer.

OBJECTIVE: To describe a micromanipulation-electrofusion procedure for transferring germinal vesicles (GVs) between immature human oocytes. DESIGN: Pilot study to assess oocyte maturation after an invasive micromanipulation procedure. SETTING: Research laboratory at a university medical center. PATIENT(S): Immature oocytes were discarded from intracytoplasmic sperm injection (ICSI)-IVF cycles of patients 23-48 years of age. INTERVENTION(S): Initially, GV removal and transfer were performed on the same oocyte; these "self-reconstructed" oocytes were then cultured in vitro for up to 50 hours and examined periodically for maturation as judged by the extrusion of the first polar body. In a second study, GVs from oocytes of "old" patients (>38 years old) were successfully transferred into enucleated immature oocytes of "young" patients (<31 years old). MAIN OUTCOME MEASURE(S): Extrusion of the first polar body was monitored in "reconstructed" and control oocytes; karyotypes also were analyzed at meiosis II. RESULT(S): From 48 oocytes from old patients, 12 GVs were successfully removed, transferred, and fused into previously enucleated oocytes from young patients. After in vitro culture, 7 of these "reconstructed" oocytes matured to meiosis II, a maturation rate not significantly different from that observed in nonmanipulated controls. A normal, second meiotic metaphase chromosome complement was observed in 4 of 5 reconstructed oocytes. CONCLUSION(S): Normal meiosis can occur after the transfer of a GV into an enucleated host oocyte. Germinal vesicle transfer may be a valuable research procedure that generates cell models to characterize the cytoplasmic-nuclear interplay for cell cycle regulation, maturation, and fertilization in the human oocyte; it also may be a potentially attractive alternative to oocyte donation.