Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: short communication

Although strategies for in-vitro maturation of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes, full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.     

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.     

Embryo development and pregnancies from in-vitro matured and fertilized human oocytes.

There is an increasing interest in retrieving immature oocytes in the absence of or with limited gonadotrophin exposure, with the aim of maturing them in vitro for embryo transfer purposes. The aim of this report is to present our experience of fertilization, embryonic development and pregnancies from in-vitro maturation cycles. A total of 18 patients underwent 21 cycles in which an average of 8.1 immature oocytes was retrieved after limited exposure to human menopausal gonadotrophin (HMG) and no exposure to human chorionic gonadotrophin (HCG). In one cycle, no oocytes were recovered. The oocytes were cultured for 44 h and 121 oocytes which reached MII were injected with a single spermatozoon. A total of 71 oocytes showed two pronuclei and 53 zygotes cleaved. Forty-four embryos were transferred in 17 cycles. Five weeks after embryo transfer, ultrasound examination indicated the presence of one gestational sac and one fetal heart beat in two patients. The results suggest that in-vitro matured oocytes can undergo fertilization and the resulting embryos may result in pregnancies. However, the success rate was not sufficient to recommend widespread use of the technique without further research.     

Fertilization and early embryology: The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme

The cumulative embryo score system involves three aspects ofrelevance in pregnancy achievement during in-vitro fertilization(IVF) and embryo transfer: cleavage rates, morphological qualitiesand the number of embryos transferred. The scores of 602 IVF/embryotransfer trials were calculated and analysed to determine thesystem's relationship to pregnancy rate, pregnancy outcome andthe incidence of twin and triplet pregnancies. The system wasalso applied to cycles where endotoxins were either presentin or absent from culture medium, in order to evaluate its validityin quality control analyses. Pregnancy rates were found to increasefrom 4%, with scores between 1 and 10, to 35% in the 41–50group. The score of 20 was the criterion for separating patientsinto poor and good pregnancy prognosis groups (P = 0.00001).Biochemical abortions occurred more frequently with scores <20(P = 0.00978), but a similar relationship was not found in clinicalabortion rates (P = 0.62206). Birth rates below and above ascore of 20 (2.8 and 19.2%, respectively) differed significantly(P = 0.0005). The scores of twins overlapped extensively withthose of singleton births, but those of all triplets were >40. The system did not reflect a correlation between embryoquality and the presence of endotoxins in culture medium.     

Aspiration of oocytes for in-vitro fertilization

An aspiration system, incorporating a regulated vacuum pump,was used to examine, in vitro, some factors that may affectoocyte collection. In an open aspiration system, as the lengthof the needle was increased, or the internal diameter decreased,the velocity (and flow rate) of aspirated fluid decreased. Therewas a difference, however, between experimental flows and thosepredicted by Hagen-Poiseuille's Law. Upon application of vacuumto a closed aspiration system, employing isolated bovine ovaries,there was an initial rapid increase in the collection tube vacuumto 85% of the selected pump vacuum followed by a more gradualrise to 100%. The vacuum within the needle similarly rose rapidlyto approximately half the selected vacuum, while the vacuumat the needle tip was ~5% of selected vacuum. The vacuums throughoutthe system briefly equilibrated as maximum flow/velocity wasreached. Flow/velocity slowed dramatically as the follicle collapsed,and stopped as the needle tip was blocked. If vacuum was maintainedduring the withdrawal of the needle from the follicle, therewas a dramatic forward flow of fluid toward the collection tube.The morphological appearance of bovine cumulus after in-vitroaspiration was generally unaltered by vacuums commonly utilizedin oocyte collection, providing the cumulus was regular, compactand refractile. The cumulus was less resistant to aspirationif it was damaged or had degenerated. These results suggestthat an intact cumulus may offer protection during ooocyte collection.     

Chromosomes of oocytes failing in-vitro fertilization

Of 263 oocytes that failed to fertilize after in-vitro fertilizationand were sent for cytogenetic investigation, 179 (68.1 %) wereanalysable. More than 72.0% were normal metaphase II haploids(23,X). Hyperhaploidy, hypohaploidy and complex cases made upa total aneuploidy rate of 12.3%, while 10.1% were diploid.In addition, there were five oocytes with structural aberrationsand eight yielded chromatids only. The total chromosome aberrationrate was 29.1%. No significant difference was found betweenthe aneuploidy rate and maternal age. Here we present photographicvidence of oocytes with extra whole chromosomes and extra singlechromatids. We suggest that both predivision and nondisjunctioncontribute to the formation of trisomy in man.     

Fertilization and early embrology: Atypical maturation of oocytes of strain I/LnJ mice

The maturation of strain I/LnJ oocytes was compared to oocytesof selected inbred strains. The time of germinal vesicle breakdown(GVB) of I/LnJ oocytes was greatly delayed compared to all otherstrains tested. In addition, 5% of the cumulus cell-enclosedoocytes isolated from antral follicles of I/LnJ mice failedto undergo GVB in vitro and 58% of the oocytes that underwentGVB failed to progress beyond metaphase I. Similar defects inthe progression of meiosis occurred when maturation was stimulatedin vivo by the administration of exogenous gonadotrophins. Whenin-vitro matured metaphase II oocytes were selected for in-vitrofertilization, similar percentages of I/LnJ oocytes underwentfertilization and cleavage to the 2-cell stage as oocytes fromanother inbred stain, C57BL/6J, and similar percentages of 2-cellstage oocytes completed the 2-cell stage to blastocyst transitionin vitro. However, unlike C57BL/6J oocytes, a much lower percentageof oocytes that matured in vivo in response to exogenous gonadotrophinsunderwent fertilization and cleavage to the 2-cell stage thanoocytes that underwent maturation in vitro. Likewise, lowerpercentages of 2-cell stage embryos derived from in-vivo maturedI/LnJ oocytes developed to blastocysts than embryos derivedfrom in-vitro matured oocytes. These results show than I/LnJoocytes are atypical in the progression of both nuclear andcytoplasmic maturation. These defects may account for the poorreproductive performance of I/LnJ mice. Thus, I/LnJ mice mightbe a useful model for studying infertility resulting from defectiveoocytes.     

Implantation: Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers

The purpose of this study was to devise an embryo score to predictthe likelihood of successful implantation after in-vitro fertilization(IVF). Unlike most studies dealing with the influence of embryostage and morphology on pregnancy, our study was based on singlerather than multiple embryo transfers. A total of 957 singleembryo transfers were carried out. No delivery was obtainedafter any of the 99 transfers using 1-cell embryos or embryosobtained after delayed fertilization. In the remaining 858 transfers,the embryos had cleaved. Higher pregnancy rates were obtainedwith embryos displaying no irregular cells (11.7 versus 6.9%;P < 0.01) and embryos displaying no fragmentation (11.5 versus8.1%; P < 0.05). The 4-cell embryos implanted 2-fold moreoften than embryos with more or less cells (15.6 versus 7.4%;P < 0.01). Based on these observations, we devised a 4-pointembryo score in which embryos are assigned 1 point each if they(i) are cleaved, (ii) present no fragmentation, (iii) displayno irregularities, and (iv) have four cells. Both pregnancyrate and take home baby rate were significantly correlated withembryo score. Each point of this score corresponds to a 4% increasein pregnancy rate. Interestingly, pregnancy rate was significantlylower in women aged >38 years (8.2 versus 11.4%; P < 0.05),even though embryo quality was similar regardless of age. Singleembryo transfer allowed us to define a simple and useful embryoscore to choose the best embryo for transfer to optimize IVFand embryo transfer outcome. The use of this embryo score coulddecrease multiple pregnancies after multiple embryo transfers.     

Incomplete denudation of oocytes prior to ICSI enhances embryo quality and blastocyst development

BACKGROUND: Granulosa cells are essential mediators of oocyte maturation and fertilization. Because of the denudation of oocytes in preparation for ICSI, any potential positive effect of surplus cumulus cells (CCs) on further development would be unable to exert further effect. In order to evaluate the actual influence of adhering cumulus cells on further preimplantation development, this prospective study was carried out. METHODS: Sibling cumulus-oocyte complexes for 57 ICSI patients were split into a study group (incomplete denudation, n = 314) and a control group (complete denudation, n = 336). According to the cumulus cell pattern after partial denudation, mature gametes from the study group were further subdivided into type A oocytes, which showed several prominent CC clusters (n = 202), and type B (n = 75), which showed a more homogeneous pattern with CC covering the whole surface of the gamete. RESULTS: In immature oocytes, presence of adhered CCs led to a significant increase in resumption of meiosis (P < 0.01). Fertilization rate (P < 0.05) and ability to cleave (P < 0.01) was impaired in the study group, because of difficulties in ICSI of type B oocytes. By contrast, embryo morphology on days 2 (P < 0.01) and 3 (P < 0.05), as well as blastocyst formation, was better (P < 0.05) in the study group (55 blastocysts out of 88 zygotes) as compared to that in the control group (49/105). CONCLUSION: These data indicate that co-culture of oocytes with attached CCs may enhance preimplantation development.     

The developmental potential of mouse oocytes matured in serum-free culture conditions

Studies were undertaken to identify serum-free conditions forthe maturation of mouse oocytes in vitro. Oocytes were recoveredfrom the antral follicles of juvenile mice 48 h after injectionwith gonadotrophin and allowed to resume meiosis in modifiedHam's F-10 (mHF-10) medium unsupplemented or supplemented withbovine serum albumin (BSA), fetal calf serum, human pre-ovulatoryserum, human follicular fluid or EDTA. They were inseminated14–16 h later, scored for polar body extrusion after 4–6h with spermatozoa, and transferred to protein-free mHF-10 forfurther development. In-vivo matured ova were inseminated andcultured in parallel as controls. Fertilization and developmentwere scored as two cells 24 h after insemination and blastocysts4 days following insemination respectively. Surprisingly, 41%of oocytes cultured in unsupplemented mHF-10 completed meiosisI, and of those, 50% fertilized; serum supplementation did notimprove maturation or fertilization rates. Although the additionof human follicular fluid to the mHF-10 improved meiosis (69%)and fertilization (68% of eggs with polar bodies) to levelscomparable with the in-vivo control eggs (79 and 66% respectively),BSA supplementation was equally beneficial. Blastocyst developmentvaried, but within each maturation/ fertilization group, thedevelopment from in-vitro matured eggs was comparable with embryosfrom in-vivo matured eggs. In addition, two out of eight 4-cellembryos from oocytes cultured in mHF-10 with BSA and EDTA gaverise to apparently normal pups following transfer to pseudopregnantrecipients. Thus, gonadotrophin-stimulated mouse oocytes cancomplete meiosis and fertilize in culture in the absence ofserum or follicular fluid. Oocytes cultured overnight in mHF-10,supplemented with EDTA and BSA, complete meiosis I, fertilizeand develop to blastocysts at rates comparable with eggs maturedin vivo. Serum-deprived oocytes have the potential to give riseto live offspring.     

Successcul embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes

In-vitro fertilization experiments with frozen/thawed rabbitoocytes were performed to develop an effective technique tobe used for the in-vitro fertilization of cryopreserved humanoocytes. Ovulatory oocytes, colected from the oviduct of virgindoes 13 h after induction of ovulation by HCG injection, werecryopreserved slowly to –30°C and plunged directlyinto liquid nitrogen. A mixture of 1.5 M 1, 3-propanediol and0.1 M sucrose was used as a cryo-protectant. After thawing,the oocytes were incubated with in-vitro capacited sperm for5 h indefined Brackett's medium. Fertilized ova were culturedfor an additional 20 h until the 4-to-8-cell stage was reached.These embryos were transferred to pseudopregnant recipient rabbitswhich were ’asychronous‘ in the sense that theyhad been given an injection of HCG 30, 24 and 18 h before startingto do the embryo transfer. A 32% survival rate of frozen/thawedoocytes was achieved. The fertilization rate was 74% (181/264)in this study. A total of 53 embryos was transferred to theoviducts of six recipients of three different asynchronicityand four young were born. The highest implantation rate (includingresorptions) of 18% could be achived in this investigation byusing –6 h asynchronous recipients, While the overallimplantation rate was 9.4 %     

The effect of propofol anaesthesia on oocyte fertilization and early embryo quality

Propofol, frequently used for i.v. induction of anaesthesia in assisted reproduction procedures, has been suspected of damaging oocytes. Concentrations of propofol have recently been shown to increase in follicular fluid during oocyte retrieval. Our study was designed to assess whether exposure to increasing concentrations of propofol has a measurable effect on in-vitro fertilization, cleavage and embryo development. A cohort of 130 women underwent i.v. anaesthesia using propofol and fentanyl. Time of anaesthesia from i. v. injection of propofol was measured, as were the doses of the two drugs. In 32 women expected to have more than 15 oocytes retrieved, first, middle and last oocytes were cultured separately. The mean time from i.v. injection to first follicle aspiration was 200 s. The mean time for the aspiration of each additional oocyte was 17.6 s. In 10 out of 11 cases where follicular fluid concentrations of propofol were measured, there was an increase from the first to the last follicle, but no difference was found in the ratio of mature to immature oocytes. Nor were any differences found in fertilization, cleavage and embryo cell number. In so far as in-vitro development reflects embryo quality, we conclude that the time elapsed between retrieval of the first and last oocyte does not affect oocyte quality.     

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation

BACKGROUND: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human. METHODS: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I. RESULTS: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control. CONCLUSIONS: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.     

Cigarette smoking and outcomes of in-vitro fertilization and embryo transfer: a prospective cohort study.

A prospective cohort study of 222 consecutive couples undergoing 297 cycles of in-vitro fertilization and embryo transfer (IVF-ET) was conducted to evaluate the impact of cigarette smoking in males and females. Compared with non-smokers, females smoking at the time of treatment had more previous pregnancies (1.16 versus 0.63, P less than 0.001), consumed more coffee per day (3.29 versus 1.85 cups, P = 0.001) and were less likely to hold a professional or skilled job (41% versus 66%). There was no difference in the response to ovarian stimulation in terms of the duration and dose of human menopausal gonadotrophin, peak oestradiol level or number of oocytes retrieved. The fertilization rate was actually higher in heavy smokers than in non-smokers (79.3% versus 61.3%, P = 0.007). The rate of embryo cleavage was retarded in a dose-dependent fashion. In smokers of 1-14 cigarettes/day, the likelihood of transferring an embryo at greater than or equal to 4-cell stage was 0.87 [95% confidence limits (CL) 0.56-1.4] and in smokers of greater than or equal to 15 cigarettes/day, the likelihood was 0.52 (95% CL 0.31-0.88). However, evaluation of interrelated factors using logistic regression suggested that a low socioeconomic status had a greater detrimental effect on embryo cleavage rate than female smoking. No significant difference was noted in the clinical outcome following embryo transfer. A study of larger sample size is required to evaluate whether the effects of cigarette smoking are independent of socioeconomic status and other related factors and whether they result in reduced ongoing clinical pregnancy and live birth rates.     

Micromorphometry and spermatozoa binding patterns of fertilized and unfertilized human oocytes after in-vitro fertilization

Human oocytes (n = 380) from 71 in-vitro fertilization patientswere measured 18 h after insemination to find out if certainparameters of oocyte morphology could be related to fertilization.In addition, the number and distribution patterns of spermatozoabound to or within the zona pellucida of 534 oocytes were analysed.The mean diameter of the human oocyte was 167.7 ± 9.5µm and its mean volume was 2.5 x 10⁶ µm³. Therewere no significant differences in diameter between 112 fertilizedand 168 unfertilized oocytes, although they displayed differencesin the size of the perivitelline space and the zona pellucida.The age of the patients had no significant effect on the morphometryof the oocytes. Sperm binding patterns did not correlate withfertilization. The number and distribution of the spermatozoaon the surface of the zona pellucida was extremely heterogeneousand was not related to the occurrence of fertilization. Allpossible binding and distribution patterns were in the samerange in both fertilized and unfertilized oocytes. In conclusion,the micromorphometry of human oocytes and their sperm bindingpatterns were not related to the occurrence of fertilization.     

Infertility: Assessments of embryo transfer after in-vitro fertilization: effects of glyceryl trinitrate

The role of embryo transfer and its associated difficultieson the outcome of human in-vitro fertilization (IVF) were examinedusing a standardized procedure and a scoring system (embryotransfer scores 1–5). This system was used to assess anyeffects of the smooth muscle relaxant glyceryl trinitrate (GTN)on embryo transfer. Patients (n = 120) were randomized in adouble-blind manner at their first embryo transfer to receivesublingual GTN or placebo before the transfer. Retrospectiveanalysis showed that higher pregnancy rates were associatedwith uncomplicated transfers (score 1; P < 0.01). The outcomemeasures included pregnancy rate, total time of cervical manipulation(embryo transfer time) and embryo transfer score. All pregnancieshad a transfer score of 1 or 2, but no recorded parameter differentiatedbetween pregnant or non-pregnant cycles, and GTN had no significanteffect on any parameter.     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

Fertilization and early embryology: Polyamines may increase the percentage of in-vitro fertilized murine oocytes that develop into blastocysts

This study was designed to investigate whether the polyaminesspermine, spermidine and putrescine can prevent the arrest ofdevelopment of in-vitro fertilized murine oocytes from CD1 strainmice. Development in M16 or Brinster's medium with or withoutpolyamine supplementation was assessed and the ability of blastocyststo attach and grow out on laminin was compared with in-vivo-generatedembryo blastocysts. Oocytes from old (20 weeks) females showedlower rates of fertilization and subsequent development in comparisonwith oocytes from young (6–8 weeks old) females, but inboth cases <5% of in-vitro fertilized oocytes achieved theblastocyst stage. In-vitro fertilized young oocytes showed enhanceddevelopment to the blastocyst stage with the addition of thepolyamine putrescine, although lesser enhancement was seen withspermine and spermidine; this effect was only evident when theoocytes were grown in Brinster's medium, reflecting the needfor a particular set of culture conditions for growth-enhancingagents to be effective. Blastocysts generated in putrescine-supplementedBrinster's medium were able to implant on laminin-coated surfacesin vitro and to respond to the cytokines colony-stimulatingfactor-1, epidermal growth factor and platelet-derived growthfactor with increased trophoblast outgrowth to a greater degreethan in-vivo-generated blastocysts. Murine in-vitro fertilizedembryo development was not enhanced by co-culture with Verocells. Putrescine was the most effective polyamine, enhancingthe in-vitro fertilized oocyte development to blastocysts ina permissive environment.     

Shortened exposure of oocytes to spermatozoa improves in-vitro fertilization outcome: a prospective, randomized, controlled study.

A prospective, randomized study of 158 patients undergoing in-vitro fertilization (IVF) and embryo transfer was conducted to evaluate whether a shortened exposure of oocytes to spermatozoa enhances oocyte development, and subsequently influences the IVF outcome. A comparison was made between conventional treatment time and shorter exposure of retrieved oocytes to spermatozoa. Fertilization and cleavage rates, embryo quality, implantation and pregnancy rates in the study group (short exposure) versus controls (standard IVF procedure) were evaluated. Fertilization (56 versus 61%) and cleavage rates (96 versus 92%) were similar in the two groups respectively. However, embryo quality was significantly higher in the study group (P < 0.05). Moreover, the pregnancy and implantation rates were significantly increased (42.4 versus 26% per embryo transfer, and 16 versus 10% respectively; P < 0.05). Our results demonstrated that shorter exposure of oocytes to spermatozoa is superior to the standard time in IVF and may have a favourable effect on implantation rates by improving embryo quality.