Fertilization and early embryology: Embryo quality and pregnancy potential of fresh compared with frozen embryos--is freezing detrimental to high quality embryos?

To determine the effect of cryopreservation on embryo qualityand the pregnancy potential of embryos, donated oocytes fromthe same donor (n = 24) were randomly allocated, with subsequenttransfer to two or more different ovum recipients resultingin at least one fresh and one frozen embryo transfer cycle fromthe same cohort of oocytes. Endometrial receptivity was controlledin all ovum recipients, and male factor patients were excluded.The number of embryos transferred, mean embryo grade transferred,number of high quality embryos (grade 2.5, grade 1 being best)transferred and embryo implantation and live birth rates arereported. Significantly more embryos (4.4 ± 1.2 versus3.3 ± 1.2, P < 0.00003) of higher quality (1.9 ±0.5 versus 2.1 ± 0.5, P < 0.013) and of a more advancedcell stage (3.0 ± 0.6 versus 2.6 ± 0.7, P <0.019) were transferred fresh than after cryopreservation respectively.Implantation rates/embryo [19/151 (12.6%) and 9/111 (8.1%)]and live birth rates/transfer [11/42 (26.2%) and 6/45 (13.3%)],from fresh and frozen transfers respectively, were not significantlydifferent despite the larger number of high quality embryostransferred fresh. Embryo cryopreservation adversely affectsembryo quality, but does not have detrimental effects on theimplantation or pregnancy potential of high quality embryos.Because of the loss of embryos during freeze — thawingduring frozen embryo cycles, every effort should be made toattempt a fresh transfer.     

A comparative analysis of embryos derived from routine in-vitro fertilization and subzonal microinsemination

Embryos obtained from patients undergoing routine in-vitro fertilization(IVF) and embryo transfer were compared with those undergoingsubzonal microinsemination (SUZI) for male factor infertility.Overall, the proportion of cleaved embryos was significantlyhigher in the IVF group in comparison with the SUZI group at48 h post-insemination [1533 out of 1609 (95.3%) versus 776out of 952 (81.5%)]. The mean ± SD grading score of theIVF-derived embryos of 3.61 ± 0.50 was significantlybetter than that for SUZI of 2.97 ± 0.86 (P < 0.0005)at the same time. The implantation rates following the replacementof IVF or SUZI embryos at 48 h were comparable: 14.3 and 10.0%respectively. However, the IVF embryo implantation rate of 15.1%at 72 h was significantly better than that following the replacementof SUZI embryos at either 48 (10.0%) or 72 h (8.0%). The replacementof SUZI-derived embryos at 48 h resulted in significantly higherpregnancy (25.0%) and implantation rates (10.0%) than at 72h, with rates of 10.8 and 8.0% respectively. Similarly, theoverall embryo quality deteriorated following in-vitro culturefor up to 72 h. The clinical pregnancy loss rate (33.0%) washighest following the replacement of SUZI embryos at 72 h, althoughthe data were limited. It is suggested that these data indicatethat a combination of in-vitro manipulation, the injection ofmultiple spermatozoa into the subzonal space and probably thegenomic capacity of spermatozoa derived from poor-quality semenmay contribute to the poorer outcome of embryo development followingSUZI. Prolonged in-vitro culture beyond 48 h appears to be deleteriousto the development of SUZI cleaved embryos and the subsequentoutcome of treatment, and hence should be avoided.     

Comparison of a fixed and dynamic protocol for embryo replacement in an IVF/ET programme

Implantation after embryo transfer is considered a major obstadein terms of pregnancy rates after in-vitro fertilization. Aflexible approach to the date of replacement, based on the factthat the most suitable embryonic structure for proper implantationis the four- to eight-cell embryo, has been studied. One-hundred-and-twentypatients with various aetiologies of infertility were stimulatedwith HMG or combined HMG and FSH, then treated by three differentmethods of embryo replacement. In group I embryos were replacedin mothers 48 h after ovum retrieval; in group II replacementswere carried out 72 h after retrieval; and in group III replacementswere related to embryonic cleavage development. Mean levelsof oestradiol when HCG was given averaged 1301 ± 121pg/ml, 1016 ± 96 pg/ml and 1182 ± 101 pg/ml inthe three groups, respectively. There was no significant differencein the average number of embryos transferred among the variousgroups. The pregnancy rates per transfer were 21.8, 24.2 and38.7%, respectively (P < 0.001). Although more investigationis required, a dynamic approach to embryo replacement mightsignificantly improve pregnancy rates, because of improved interactionsbetween the embryos and the uterus.     

Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells

A study was undertaken to evaluate embryonic development andestablish pregnancies with human embryos after in-vitro culturein two different systems. Treatment A consisted of culturingzygotes in serum-supplemented human tubal fluid culture medium(HTF). Treatment B consisted of culturing zygotes on a monolayerof bovine oviductal epithelial cells with HTF. At the time ofembryo replacement, embryos in treatment B had 4.11 blastomerespresent, which was greater (P < 0.05) than the 3.81 presentfor embryos in treatment A. In addition, the cellular fragmentationrate for treatment A embryos was 1.10, which was greater (P< 0.05) than the fragmentation rate of 0.38 for embryos withintreatment B. The incidence of ongoing pregnancy was higher afterreplacement of co-cultured embryos (treatment B) (43%) thanreplacement of conventionally cultured embryos (treatment A)(29%). The implantation rate per embryo increased (P < 0.05)from 11.5 to 18.4% after co-culture. In treatment B the proportionof ‘spare’ embryos developing to expanded blastocystswas 58.5%, which was greater (P < 0.05) than the blastocystdevelopment rate of 29.3% observed for embryos within treatmentA.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

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.     

Tubal embryo transfer in cynomologus monkeys: effects of hyperstimulation and synchrony

To date the main limitation of in-vitro fertilization–embryotransfer (IVF–ET) programmes is that transcervical transferof embryos results in a rate of low implantation. On the otherhand, the technique of gamete intra-Fallopian transfer (GIFT)does not contribute to information on oocyte fertilization rates,and the time of oocyte exposure to sperm may be limited. Thedevelopment of ultrasonically guided follicular aspiration willallow transfer of embryos generated in vitro to the Fallopiantubes performing only one surgical procedure in the process.We have perfonnd 25 intra-tubal embryo transfers in the cynomolgusmonkey (Macaca fascicularis). Ovarian stimulation, folliularaspiration and IVF procedures have been reported previouslyby Balmaceda. ETs were performed via laparotomy. Embryos atthe 2-, 4- or 9-cell stage were loaded into a tom-cat catheterin 5 ?l of culture medium and delivered to the mid-ampullaryportion of the tube. Seven ETs performed during stimulated cyclesresulted in one pregnancy, and 18 ETs performed in synchronizedrecipients resulted in six pregnancies. Ten ETs were performed0–24 h, eight performed 24–48 h and seven performed72–110 h after follicular aspiration or ovulation, andresulted in 4, 3 and 0 pregnancies respectively. Our resultsdemonstrate that intra-tubal embryo transfer can result in normalintra-uterine pregnancies and suggest that both ovarian stimulationand cycle synchronization affect the probability of embryo implantation.     

Fertilization and early embryology: A bridge to intracytoplasmic sperm injection--high insemination concentrations benefit patients who have a reduced chance of fertilization with standard in-vitro fertilization

This study was carried out to determine whether high inseminationconcentrations (HIC) could improve fertilization and pregnancyrates in patients who had either previously demonstrated poorfertilization rates in vitro using standard protocols (Group1) or in whom a reduced chance of fertilization was indicatedat semen assessment prior to in-vitro fertilization (IVF) (Groups2 and 3). Forty nine patients were recruited for the study.Standard IVF was carried out in 1 ml volumes using 10⁵ spermatozoa/mlHIC treatment involved co-culture of spermatozoa and oocytesin microdroplets with insemination concentrations increased10–50 fold higher than standard IVF. Fertilization andpregnancy rates were compared between standard IVF and HIC inindividual patients either in consecutive cycles (Group 1) orusing sibling oocytes in the same cycle (Group 2). Group 3 patientswere treated with HIC for their first treatment cycle. HIC significantlyimproved the fertilization rate compared with standard IVF forGroups 1 (59.7±10.7 versus 19.6±5.4% respectively)and 2 (54.9±8.5 versus 34.0±85% respectively).HIC increased the pregnancy rate from 0% with standard IVF to20% per embryo transfer in Group 1 patients. A single pregnancyderived from the transfer of HIC and IVF embryos occurred inGroup 2. The fertilization rate (47.2±7.6%) and pregnancyrate (31.3% per embryo transfer) for Group 3 patients was higherthan predicted. There was no increase in the rate of polyploidywith HIC. Provided there are sufficient numbers of motile spermatozoa,HIC may be offered as an initial form of treatment, thus permittingreferral of only the poorest responders for intracytoplasmicsperm injection (ICSI).     

Preimplantation adoption: establishing pregnancy using donated oocytes and spermatozoa

The experience of transferring embryos produced through in-vitrofertilization (IVF) utilizing donated oocytes and spermatozoais described. Recipients (n = 28; aged 38–59 years) receivedoral micronized oestradiol and i.m. progesterone and were synchronizedto donors undergoing ovarian stimulation. Reasons for selectingtherapy included advanced reproductive age (>42 years; n= 21) or hyper-gonadotrophic hypogonadism (n = 7), combinedwith severe male factor infertility in 23 couples. Five womenwere single and without partners. Oocytes were fertilized bycryopreserved spermatozoa designated for use by the recipient.Up to five embryos were transferred trans-cervically. Supernumeraryembryos were cryopreserved. A total of 36 aspirations produced15.6 ± 7.3 oocytes per retrieval. In 10/36 cycles (27.8%),embryos were available for cryopreservation. Using fresh embryos,the overall pregnancy rate was 38.9% (14/36), clinical pregnancyrate 33.3% (12/36), and ongoing/delivered pregnancy rate 30.6%(11/36). Three ongoing pregnancies were later established bytransferring cryopreserved embryos. Adjusting for these events,the per aspiration overall pregnancy rate per retrieval was47.2%, clinical pregnancy rate 41.7%, and ongoing/deliveredpregnancy rate 38.9%. Implantation rates per individual embryotransferred were 16.6% following fresh embryo transfer. A viablepregnancy was achieved by 14 of 28 women (50% cumulative pregnancyrate). We conclude that using donor oocytes and donor spermatozoais efficacious and allows couples of whom both members sufferfrom severe gamete abnormalities and single functionally agonadalwomen an effective means of achieving pregnancy.     

Comparison of concurrent pregnancy rates for in-vitro fertilization-embryo transfer, pronuclear stage embryo transfer and gamete intra-Fallopian transfer

Concurrent pregnancy and implantation (sacs/embryos transferred)rates were compared for 84, 77 and 49 cases of in-vitro fertilization–embryotransfer (TVF–ET), pronuclear stage embryo transfer (PROST)and gamete intra-Fallopian transfer (GIFT), respectively. Allcases reported occurred during an 18-month interval since theinitiation of PROST by our programme. Leuprolide acetate wasused with follicle stimulating hormone and human menopausalgonadotrophin for follicular stimulation of all but donor oocytecases (n = 9). Clinical pregnancy (per transfer) and implantationrates were significantly higher (P < 0.03) for PROST (52.4%,20.2%) in comparison with IVF–ET (26.9%, 11.4%). Ratesfor GIFT (48.9%, 18.4%) were not significantly higher (P = 0.10,0.14) than for IVF—ET. This was probably due to the lowernumber of GIFT than PROST procedures performed. The total pregnancyrate for GIFT (biochemical, ectopk and clinical combined) wassignificantly greater (P < 0.05) than for IVF—ET. Pregnancyand implantation rates for PROST and GIFT were similar. Theseresults support the use of PROST rather than IVF—ET forall cases in which the woman has one functional Fallopian tube.Furthermore, to maintain equivalent rates of pregnancy withPROST and GIFT, it is suggested that GIFT should not be usedfor cases of male-factor infertility without first documentingnormal rates of in-vitro fertilization with PROST.     

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation

It has been shown recently that delayed transfers improve implantationrates in assisted reproductive technology programmes. In a prospectivestudy, the pregnancy rates and safety of outcome were evaluatedin a group of patients after the transfer of day 5 blastocystswith enzymatic treatment of the zona pellucida. Nineteen womenwith a mean age of 32.6 ± 5.2 years and mean 2.1 ±2.2 repeated attempts had blastocyst transfers with a mean numberof 2.5 ± 0.7 embryos replaced per patient. The clinicalpregnancy rates per cycle/transfer and implantation rate were53% and 33%, respectively. The multiple pregnancy rate was 40%(two pregnancies were triplets). The pregnancy and implantationrates were very much higher than observed for most assistedreproduction technology centres. The ‘in-vitro implantation’rates of zona-free blastocysts on a variety of feeder monolayerswas 92%, offering some thoughts as to the role of the zona andinteraction of the inner cell mass and trophoectoderm with theendometrium in implantation. Based on the in-vitro studies andthe high multiple pregnancy rates, it appears that zona-manipulatedblastocysts implant relatively well and there would be a needto reduce the number of transferred embryos to one or two, thusreducing multiple pregnancies and having spare blastocysts availablefor cryopreservation. The results also suggest that using theembryo culture protocol and method of transfer in the presentstudy offers encouraging improvements to assisted reproductiontechnology, and enzymatic treatment of the zona may allow betteranchorage and dialogue of the embryo with the endometrium, helpingus to improve and understand implantation.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Efficacy of the sperm survival test for the prediction of oocyte fertilization in culture

The present study was carried out to investigate the predictivevalue of the sperm survival test (SST) with respect to the fertilizationof oocytes in culture. In general, our laboratory uses a totalof 50 000–150 000 motile spermatozoa to inseminate eachoocyte. The remaining material is evaluated for motility beforeand after 24 h of incubation at 37°C in a 5% CO₂ atmosphere.A total of 250 oocytes from 50 cases (mean ± SD, 5.0± 2.4 oocytes per retrieval) were inseminated and thefinal rate of cleaved embryos obtained was 52.5%. The SST (%)was considered normal when the ratio (final density of progressingspermatozoa after 24 h x 100/initial density of progressingspermatozoa) was 50% or more. Any other result was consideredabnormal. Cases presenting one or more cleaved embryos (n =40) were separated from those in which no embryo formation occurred(n = 10) and the results were compared in terms of the respectivesperm survival rates over a period of 24 h: normal SST (oneor more cleaved embryos, 37; none, five), abnormal SST (oneor more cleaved embryos, three; none, five). The specificityof the SST was 0.92 and sensitivity 0.50, the predictive valueof the abnormal test was 0.62 and the predictive value of thenormal test 0.88. The efficacy of the test was estimated at0.71, which was better than the conventional parameters of spermanalysis. A receiver — operating characteristics curvefor SST confirmed that the test can be useful for the predictionof fertilizability of oocytes in the laboratory.     

Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization

The impact of intracytoplasmic sperm injection (ICSI) on cryopreservedzygotes and embryos was evaluated by comparing embryo survivaland implantation between embryos derived from ICSI and thosederived from standard insemination procedures. The study includedpatients whose excess zygotes and embryos were cryopreservedbetween September 1993 and December 1994 and who subsequentlyunderwent a frozen embryo transfer. Embryo survival, clinicalpregnancy rates per transfer and pregnancy outcome were compared.Three hundred and thirty eight cryopreservation cycles, duringwhich 1471 embryos were cryopreserved, were included in thisstudy. Of those, 961 were derived from oocytes fertilized byinsemination in vitro and 510 were derived from oocytes fertilizedby ICSI. A total of 690 of the embryos (451 in the inseminationgroup and 239 in the ICSI group) have since undergone a thawcycle. The embryo survival rates were similar between the twogroups (70.5 and 73.2%, insemination and ICSI respectively)and were not significantly affected by the stage at cryopreservation.There was no significant difference in pregnancy rates per transfer(31.8 and 32.3%), the preclinical pregnancy loss rate (16.7and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%)between the insemination and the ICSI groups respectively. Itis concluded that ICSI does not have an adverse impact on thesurvival and successful implantation of cryopreserved and thawedembryos.     

Fertilization and early embrology: Vero cell effect on in-vitro human blastocyst development: preliminary results

Asynchrony between embryo and uterine environment is one ofthe major limits in human in-vitro fertilization (TVF). A culturesystem which could prolong culture time and increase embryoniccleavage rate and viability would improve success rates. UsingVero cells, an in-vitro co-culture system was developed to investigateand promote human embryo development. Vero cells provide goodsupport for human early embryos up to the blastocyst stage.When fertilized embryos were co-cultured, 68% of them reachedthe blastocyst stage. Pregnancy rate was 50% per transfer inpatients with several previous failures of implantation. A significantincrease in clinical pregnancy rate was also demonstrated whenzygotes were maintained on Vero cell monolayer for only 24 h.The beneficial effect of the feeder layer may be through therelease of embryotrophic factors and the detoxification of theculture medium by the cells. Co-culture is a new concept inassisted reproduction.     

Relationship between the threshold of ovarian sensitivity to human menopausal gonadotrophin stimulation and in-vitro fertilization treatment outcome

In a retrospective study of 813 oocyte retrieval–embryotransfer cycles in women with normal follicle stimulating hormoneand luteinizing hormone concentrations, we sought to investigatethe relationship between the amount of human menopausal gonadotrophin(HMG) used for ovarian stimulation and treatment outcome. Patientswere divided into three groups: group A patients (495 cycles)required <40 ampoules of HMG and had a predicted probabilityfor pregnancy of 25% per embryo transfer; group B patients (165cycles) required 41–77 ampoules per cycle, with a predictedprobability rate for pregnancy of 5–25% per embryo transfer;and group C patients (153 cycles) required >77 ampoules ofHMG and the predicted probability for pregnancy was <5% perembryo transfer. Groups C and A differed significantly (P <0.005). The mean oestradiol concentration on the day of HCGadministration in group C was 6412 pmol/l, and the mean numberof eggs retrieved was seven. The highest success rates werefound when up to 2.5 ampoules of HMG were required for eachegg or 4.4 ampoules for each embryo. The lowest rates were obtainedwhen >4.8 ampoules of HMG were necessary for each oocyteor >9.6 ampoules for each embryo (P < 0.005). We identifieda group of infertile patients who required excessive amountsof HMG to achieve a fair degree of steroidogenesis, number ofeggs and number of embryos but who had very low pregnancy rates.Although all other relevant parameters were normal, this mayhighlight the beginning of ovarian–gamete insufficiencybefore the basic hormonal status is affected. In cases of repeatedfailure, oocyte donation should be considered.     

Cryopreservation of human embryos and oocytes

The success rate of human embryo cryopreservatlon depends ontechnical and embryonic parameters. First of all, the cryoprotectantcan affect embryo survival as we found by comparing two freeze-thawprocedures using propanediol (PROH) (1.5 mol) alone or withsucrose (0.1 mol). Embryo survival was significantly enhancedwith sucrose (62 versus 32%). Embryo quality is another majorparameter involved in the success of freezing; the rates ofpositive survival were found to be 67% for morphologically normalembryos versus 49% for embryos with fragments (P < 0.001).The efficiency of embryo cryopreservatlon in an IVF programmecould be estimated in 1986: a woman with extra embryos, storedafter transfer of 3–4 fresh embryos (16% of all cydes),can expect a 22% pregnancy rate per transfer of fresh embryosand a 32% pregnancy rate per collection after transfer of thestored eggs. A comparative study of the cryopreservability ofimmature or mature oocytes was performed in humans. Human oocyteshave a low survival rate (36%) whatever the cryopreservationprotocol or the initial maturation stage. Immature human oocytescould survive freezing and thawing, mature and be fertilizedin vitro, but with a very low efficiency.     

Pregnancy: Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in-vitro fertilization

Premature luteinization has been reported to be associated withdecreased pregnancy rates in patients undergoing in-vitro fertilization.However, the detrimental effect created by a pre-aspirationrise in progesterone is difficult to assess since ovarian stimulationaffects both oocyte quality and endometrial receptivity. Therefore,the relationship between premature luteinization and pregnancyrates remains uncertain. To achieve improved control for confoundingvariables, we studied premature luteinization in ovum donorsof proven fertility. A total of 114 consecutive ovum donationcycles using pituitary suppression with a gonadotrophin-releasinghormone agonist followed by gonadotrophin stimulation were examined.Serum progesterone concentration on the day of administrationof human chorionic gonadotrophin (HCG) was > 1.2 ng/ml in29% of patients. Patients were divided into two groups basedon this value. There was a significant increase in clinicalpregnancy rates per embryo transfer in the group with higherprogesterone concentrations (53 versus 25%, P = 0.012), as wellas significantly more oocytes obtained at aspiration (19.6 ±10.4 versus 13.3 ± 5.4, P < 0.001), and significantlyhigher peak serum oestradiol values (3903 ± 1787 versus2453 ± 1232 pg/ml, P < 0.001). There were no significantdifferences between groups due to age, degree of stimulationor the number of embryos transferred. We conclude that prematureluteinization as based on elevated serum progesterone concentrationis a common occurrence in oocyte donors, reflects healthy folliculardevelopment, and is associated with increased pregnancy rates.     

In-vitro development of in-vivo produced rhesus monkey morulae and blastocysts to hatched, attached, and post-attached blastocyst stages: morphology and early secretion of chorionic gonadotrophin

The earliest time of secretion of chorionic gonadotrophin (CG)by primate embryos and its role during preimplantation developmentand implantation are not clearly determined. We cultured in-vivofertilized/developed zona-intact, morphologically normal morulae(n = 11) and early blastocysts (n = 11), freshly recovered (bynon-surgical uterine flushing) on days 5 and 6 of pregnancy,respectively (day 0 = the day following LH surge), from non-superovulatednaturally bred rhesus monkeys (Macaca mulatta). Embryos werecultured for a minimum of 24 days in dishes containing 1 mlof CMRL-1066 supplemented with 20% bovine fetal serum in a humidifiedatmosphere of 5% CO₂ in air at 37°C. The culture mediumwas changed every 48 h. The percentage of hatched blastocysts,developed from morulae and early blastocysts, was 90.9; elapsedtimes (mean ± SEM) were 67.8 ± 4.4 h (morula)and 37.8 ± 3.6 h (blastocyst). The minimum number ofHoechst-stained cells/hatched blastocyst was 531. The mean diameter(± SEM) of cultured embryos increased from 180 µmat the beginning of culture to 374 ± 28 and 450 ±19 µm at the fully expanded and hatched blastocyst stages,respectively. Hatched blastocysts continued to expand (maximumdiameter: 1125 ± 25 µm); after an additional 94–96h they attached firmly to the serum-coated dishes and producedhighly proliferating multinucleate trophectodermal cells, extendingto a maximum diameter of 2–6 mm by 11–21 days ofculture. Biologically active CG in embryo-grown, serial spentmedia samples was measured in a mouse Leydig cell bioassay.The embryonic secretion of CG (ng/ml, mean ± SEM) commencedjust prior to hatching ( 0.014 ± 0.0), increased to 1.7± 0.5 after hatching but prior to attaching, and to 122.7± 45.5 by 5–11, 5108.7 ± 1706.0 by 10–17days, and decreased to 317.0 ± 201.4 by 16–40 daysin culture. These results show firstly that in-vivo producedrhesus monkey morulae and early blastocysts develop in vitroto hatched and attached blastocyst stages, exhibiting extensivetrophectodermal outgrowths. Secondly, the secretion of bioactiveCG commences from low levels during the pre-attaching blastocyststage, and increases exponentially after the attachment andtrophectodermal outgrowth of cultured embryos.     

Pregnancy: Pregnancy and implantation rates in normal replacement versus stimulated cycles

The relative importance that endometrial development and embryoquality have on implantation rates achieved with assisted reproductivetechnology (ART) is the subject of controversy. Ovarian stimulationhas been repeatedly mentioned as having a detrimental effecton endometrial receptivity (Paulson et al., 1990, Fertil. Steril.,53, 870–874). We compared pregnancy and implantation ratesachieved with ART during stimulated cycles and hormonal replacementcycles, in patients matched for the following criteria: age<35 years for the patient donating oocytes; transfer of atleast two good quality embryos/oocytes and good quality transfer.All transfer cases performed during hormonal replacement cycleswere done with donated oocytes. Comparison of results betweentechniques was not attempted due to potential differences inpopulations. The pregnancy and implantation rates achieved witheach technique during stimulated and hormonal replacement cycleswere not statistically different. In contradiction to previousresults, our data suggest that differences in uterine receptivitybetween stimulated and hormonal replacement cycles in the agegroup studied are not of critical importance in embryo implantation.Good embryo quality appears to be the dominant factor in determiningthe success of ART.