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.     

Fertilization and early embryology: Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection

The behaviour of sperm cells after intracytoplasmic sperm injection(ICSI) was investigated by analysing 192 unfertilized and 37one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilizedoocytes were directly fixed whereas 110 were first parthenogeneticallyactivated by puromycin. In contrast to the findings in unfertilizedoocytes after in-vitro fertilization, most unfertilized oocytesafter ICSI (n = 76) contained evidence of the presence of spermatozoain the cytoplasm. Few oocytes (n = 6) contained prematurelycondensed sperm chromosomes (PCC), whereas the majority containedeither intact sperm heads (n = 31) or swollen sperm nuclei (n= 39) along with metaphase II chromosomes of the oocyte. Followingactivation by puromycin, swollen sperm nuclei and PCC were nolonger observed, whereas unchanged sperm heads persisted in12 oocytes displaying a single pronucleus. A non-decondensedsperm nucleus along with decondensed maternal chromatin werealso discovered in 32 out of 37 oocytes displaying a singlepronucleus after ICSI. The findings in unfertilized and 1PNoocytes after ICSI indicate that successful sperm injection,even followed by oocyte activation, is not sufficient to guaranteenormal fertilization. It seems that partial sperm membrane damageprior to injection is also required to ensure normal sperm decondensation.     

Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle

PROBLEM: Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. METHOD OF STUDY: In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when > or = 9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. RESULTS: Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos > or = 9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. CONCLUSION: TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos > or = 9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.     

Meiotic and cytoplasmic maturation of oocytes collected in stimulated cycles is asynchronous

Oocytes collected in stimulated cycles are more readily fertilizedafter preincubation than are oocytes inseminated immediatelyafter collection. It has not been ascertained, however, whetherthis increase in the fertilization rate is due to extrusionof the first polar body (meiotic maturation) during this period,or to some conclusive cytoplasmic maturation subsequent to thepolar body extrusion. When analysing oocytes with a polar body(n = 14) by transmission electron microscopy, differences wereobserved in the appearance of cytoplasmic features which werecorrelated to the total durations both of systemic human chorionicgonadotrophin influence before collection and of oocyte culture.These differences were manifested as a delayed formation inaggregates of the smooth endoplamic reticulum in the ooplasmof oocytes having a polar body and might have signified a cytoplasmicmaturation. The degree of synchrony in the oocytes varied andthis could explain why some oocytes can be fertilized when inseminatedshortly after collection and others not until 8 h or even moreafter collection. Thus, although meiotic and cytoplasmic maturationis likely to be synchronized at ovulation of an oocyte in anatural cycle, they appear to be mainly asynchronous in oocytescollected in stimulated cycles.     

Andrology: Micromanipulation improves in-vitro fertilization results after epididymal or testicular sperm aspiration in patients with congenital absence of the vas deferens

In all, 58 couples suffering from infertility because of congenitalbilateral absence of the vas deferens underwent a total of 67combined microsurgical epididymal aspiration or testicular spermextraction (TESE) and in-vitro fertilization (TVT) treatments.The oocytes recovered were inseminated by either the microdropletIVF technique (n=20), subzonal insemination (SUZI; n= 10) orintracyto-plasmic sperm injection (ICSI; n= 37). Of the ICSIcycles, 12 were performed using spermatozoa obtained by TESE.Fertilization rates for epididymal spermatozoa were significantlyhigher for SUZI (17.9%, 17/95) and ICSI (34.4%, 137/398) thanfor microdroplet IVF (5.2%, 18/343) cycles. The proportion ofcycles in which fertilization was achieved was higher in theSUZI (80%) and ICSI (95%) cycles than in the IVF cycles (45%).Delivery or an ongoing pregnancy was achieved in one (5%) IVFcycle, two (20%) SUZI cycles and seven (18.9%) ICSI cycles.SUZI or ICSI using epididymal or testicular spermatozoa significantlyimproved the oocyte fertility rate. The ICSI procedure was especiallyadvantageous in patients for whom spermatozoa were obtainedfrom a testicular biopsy.     

Oxygen consumption by human oocytes and blastocysts grown in vitro

Oxygen consumption by human oocytes and blastocysts was analysedusing a sensitive micro-spectrophotometric technique. Oocyteswere obtained from women undergoing lapar-otomy for sterilizationor hysterectomy, and blastocysts were obtained as spare embryosfrom the local in-vitro fertilization programme. There was nosignificant difference between oocyte oxygen consumption onthe day of isolation and after 1 day of culture, 0.53 ±0.08 (n =9) and 0.35 ± 0.06 (n =5) nl/h oocyte, respectively.Blastocyst oxygen consumption was measured after a culture periodof 5–8 days after in-vitro fertilization. Mean oxygenconsumption by healthy looking blastocysts was 0.34 ±0.03 (n =15) nl/h blastocyst, and there was no change in oxygenconsumption with culture time. This level of respiration waslower than expected, both when compared with the oocytes andwhen compared with levels reported for blastocysts of otherspecies.     

The influence of the site of sperm deposition and mode of oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates

The aims of this study were to examine, in a prospective, controlledway, the effect of the sperm deposition site in the oocyte andthe mode of oolemma breakage in intracytoplasmic sperm injection(ICSI) on fertilization and embryo development rates. In thefirst trial (100 cyclesin total), the spermatozoa were depositedfurther from themeiotic spindle (polar body at the 12 o'clockposition) in half of the oocytes (n = 649), while in the otherhalf(n = 605) the spermatozoa were deposited nearer to themeioticspindle (polar body at the 6 o'clock position). In the secondtrial (6860 oocytes in 624 cycles), five different modes ofmembrane breakage (the reaction of the oolemmato the penetratinginjection needle) at the moment of injection were noted: oolemmabreakage, type A pricking once, no suction (n = 1401); typeB, pricking once, smallsuction (n = 2761); type C, prickingonce, long suctionin = 2310); type D, pricking twice or more,no or small amount of suction (n = 259); and type E, prickingtwiceor more, long suction (n = 129). No differences were observedbetween the 12 and 6 o'clock positions in the survival rate(90 and 90% respectively) and in the normal fertilization rates(78 and 77% respectively). Significantly more transfer qualityembryos (50% fragmentation) were obtained in the 6 o'clock positiongroup (83%) than in the 12 o'clock position group (79%). Inthe second trial, significantly lower survival rates were notedafter membrane breakage type A (82%) than after breakages oftypes B, C, D and E (93, 92, 88 and 88% respectively). Therewere no significant differences present in the normal fertilizationrates (70, 72, 70, 71 and 73% for types A-E respectively), butsignificantly more freeze quality embryos (20% fragmentation)were obtained after injection B (65%) than after injection typesA, C, D and E (59, 61, 55 and 51%respectively). In conclusion,the site of sperm deposition inthe oocyte does not influencethe normal fertilization rate but does affect the embryo developmentrate. Furthermore, the mode of membrane breakage does not influencethe normal fertilization rate but does affect oocyte survivalandembryo development rates.     

Endocrinology: Predictive value of serum oestradiol concentrations and oocyte number in severe ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is a serious complicationof gonadotrophin usage but it is difficult to accurately predictits occurrence. Previous investigators have identified the combinationof high oestradiol concentrations and oocyte number as beingpredictive in 80% of cases. In this study we sought to identifythe incidence of severe OHSS in patients with high oestradiolconcentrations and large numbers of oocytes and to evaluatethe importance of pregnancy in the development of OHSS. Between1990 and 1993, we studied 139 cycles using two assisted reproductivetechniques [oocyte donor, n =72; in-vitro fertilization (IVF),n = 67] in which either oestradiol (>4000 pg/ml), oocytenumber (>25), or both were elevated. OHSS was diagnosed bystandard criteria. There were no cases of severe OHSS in theoocyte donor group and six in the IVF group. Among 10 patientswith oestradiol concentration >6000 pg/ml and >30 oocytes,only one had OHSS (10%). The relative risk of OHSS with pregnancywas 12 (confidence interval 2.18–66.14). We conclude thatthe risk of OHSS even at high levels of stimulation is lowerthan previously believed. Secondly, donors have a very low riskof OHSS, probably because of the absence of pregnancy. As such,cryopreservation of all oocytes in IVF cycles is a reasonablealternative to cycle cancellation or use of adjunctive medication.     

Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection

The aim of this study was to analyse the various reactions displayedby the oolemma to the penetrating pipette during intracytoplasmicsperm injection (ICSI) and correlate them with clinical factors,oocyte survival and fertilization patterns. Three types of oolemmaresponses were observed: normal breakage, when the injectionneedle created an invagination that ruptured at the approximatecentre of the egg; sudden breakage, when the membrane brokewithout creating a funnel; and difficult breakage, when themembrane did not break or broke after several penetration attempts.A total of 2928 oocytes were analysed with the following observations:73.9% (n = 2164) experienced normal breakage, 11.8% (n =345)sudden breakage, and 14.3% (n = 419) difficult breakage. Thesurvival rate and number of normally fertilized oocytes weresignificantly lower and the incidence of digynic oocytes wassignificantly higher in the sudden breakage group; furthermore,in this group a significantly shorter length of stimulationwas observed along with lower serum oestradiol concentrationswhen compared to oocytes experiencing normal and difficult breakagepatterns. These recorded patterns were predictive of the survivaland fertilization ability of the injected oocytes, as well asthe incidence of digyny. The link between membrane behaviourand various clinical parameters appears to indicate a correlationbetween the modality of stimulation and oolemma characteristics.     

Fertilization and early embryology: Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose

Feasibility of cryopreservalion of mature human oocytes using1,2-propanediol and sucrose was studied initially utilizrng1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes55% survived thawing, and 41% of 128 oocytes inseminated bysingle sperm intracytoplasmlc injection (ICSI) fertilized normally.Limited embryonic development occurred in 51% of these embryos(n = 27) observed for the next 4 days. Cryosurvlval of freshdonated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization(n = 13; 65%) and embryo development (100%) was good prior uterinetransfer on day 3. Eight oocyte recipient cycles were undertaken,in which cryopreserved donated oocytes were thawed and inseminatedby ICSI. Five of these cycles reached embryo transfer, and threepregnancies were initiated though none went successfully toterm. Oocyte cryopreservatlon will ultimately facilitate oocytedonation procedures; however, cryosurvival of fresh frozen oocytesmust be improved to at least the degree observed with aged unfertilizedoocytes.     

Fertilization in vitro of human oocytes by spermatozoa collected in different stressful situations

It has been shown that semen quality is impaired in couplesundergoing in-vitro fertilization (IVF), probably due to stress.A possible effect of stress on the ability of spermatozoa tofertilize human oocytes in vitro was analysed in the presentstudy composed of 26 couples with normozoospermic men undergoingIVF. A semen sample was obtained during the infertility work-upand was cryopreserved (sample 1). A second sample (sample 2)was provided after oocyte retrieval during the IVF cycle. Sample1 was thawed and both samples were washed and preincubated foroocyte insemination. One-hundred-and-five oocytes were inseminatedusing thawed sample 1, and 120 with sample 2.Semen parameterssuch as density, progressive motility and percentage of abnormalforms were compared between sample 1, before and after freezing,and sample 2. Only motility was significantly (P<0.01) decreasedby cryopreservation in sample 1, but no parameter was significantlydifferent when fresh sample 1 was compared to sample 2. Thefertilization rate was 78.6% using sample 1 in comparison to87.5% when sample 2 was employed (not significant, NS). Cleavagerates were 77.7 and 89.7%, respectively (NS). A group of fivepatients undergoing IVF who needed donor semen served as a controlfor the effect of sperm cryopreservation on IVF. In these cases,the donor was asked to provide a fresh sample. Half of thissample was frozen and thawed. Subsequently, fresh and thawedsamples were prepared for insemination and oocytes inseminatedeither with the fresh preparation (n=24) or the frozen and thawedspermatozoa (n=22). There was a significant (P<0.05) decreasein motility in the thawed sample, but fertilization and cleavagerates were not different. These data suggest that the stressfulsituation induced by IVF treatment in normozoospermic men doesnot affect the ability of spermatozoa to fertilize human oocytesin vitro. Cryopreservation of human spermatozoa before IVF maybe a good policy in couples especially suspected of being understress during this procedure.     

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation

The present study was conducted to determine if the cryopreservationof immature human oocytes has a deleterious effect on the meioticspindle following maturation in vitro. Oocytes were obtainedin excess from in-vitro fertilization patients and divided intofour groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immatureoocytes cryopreserved before or after maturation in vitro respectively.Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controlsand included oocytes matured in vitro and in vivo respectively.The meiotic spindle was identified after incubation in anti-tubulinmonoclonal antibody (1 h, 37°C) and fluorescein-conjugatedgoat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomeswere counterstained with 4‘, 6’-diamidino-2-phenylindole.Following cryopreservation, group 1 oocytes demonstrated a 63%survival rate and 68% maturation rate in vitro. In all, 58%of the oocytes in group 2 survived the thaw. The number of oocyteswith normal spindles in group 1 (81.0%) was not significantlydifferent from control groups 3 (83.8%) and 4 (88.9%), whilethe number of group 2 oocytes with normal structures (43.5%)was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002),and 4 (P = 0.025). These results suggest that cryopreservationof the prophase I human oocyte does not significantly increaseabnormalities in the resulting meiotic spindle.     

Fertilization and early embryology: Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes

Although the average fertilization rate in most in-vitro fertilization(IVF) centres is 60–70%, there are cases of complete orvirtually complete fertilization failures. The aim of our workwas to study the fertilization and the subsequent cleavage characteristicsof 1-day-old human oocytes treated by intracytoplasmic singlesperm injection (ICSI) after failing to fertilize during thestandard IVF procedure. A total of 115 metaphase II 1-day-oldunfertilized oocytes were collected from 23 patients. No additionaltreatment was applied to the oocytes or to the semen sample.A single spermatozoon from the patient's husband was injectedinto the cytoplasm of each of these oocytes 21–33 h afterovum retrieval. Injected oocytes were observed at 16–18h and again 42–44 h after the ICSI procedure. Of the injectedoocytes, 92% (n = 106) were intact after ICSI, 38% (n = 44)had two distinct pronuclei and there was no difference in thefertilization rate of oocytes when andrological and non-andrologicalpatients were compared. Similarly, there was no difference inthe fertilization rate after ICSI where patients with acceptableor good (> 15%) fertilization after standard IVF were comparedto patients who had poor (<15%) fertilization after IVF.There was no significant difference in the sperm concentrationor in the progressive forward motility (a + b motility) in thesegroups except where a + b motility of andrological and non-adrologicalpatients was compared. The majority (84%) of the normally fertilizedoocytes cleaved and most (77%) of these embryos showed <20%fragmentation 2 days after the ICSI procedure. From this studyit can be concluded that 1-day-old metaphase II oocytes whichhave failed to be fertilized after standard IVF procedure canbe fertilized and cleave when ICSI is performed on them theday after oocyte retrieval.     

Principle and practice of in vitro fertilization. Role in the treatment of female sterility

In vitro fertilization (IVF) and embryo transfer (ET) appear to constitute a revolution in the reproductive sciences rather than merely a new technique in the treatment of sterility. Principle of IVF: IVF accomplishes in vitro the process than normally occurs in the oviduct between the ovulation of oocyte II and embryo implantation in the endometrium. This 4 day period (under normal conditions in the woman) involves 4 steps: recovery, fertilization, segmentation and transport. Performance of IVF: Recovery of the oocytes: The oocytes are recovered under celioscopic or echographic observation when they have completed cytoplasmic maturation and their first meiosis. A precise monitoring of ovulation (spontaneous or induced) should be performed using estrogen and LH assays. IVF provides an opportunity for evaluating the methods of ovulation induction and monitoring, as a function of the maturation of the oocytes recovered. Fertilization: When the oocyte has achieved maturing after several hours of incubation, fertilization is obtained 15 h contact with washed and capacitated spermatozoa (100 000/ml). This step is highly dependent on gametocyte quality: oocyte maturity and fecundity of spermatozoa, which can be estimated from the percentage of survival in the insemination medium. Segmentation occurs in culture at pH 7.28 in the presence of 5 per cent CO2 at 37 degrees C (pronucleus 15th, 2 blastomeres 26 h, 4-8 blastomeres 52 h). Embryo transfer is carried out when an embryo is present at 52 h. Only 1/10 of the embryo transfers result in successful implantation, which depends on the quality of the embryo; the quality can only be indirect criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of spermagglutination with proteolytic enzymes, II. Sperm function after enzymatic disagglutination

Antibody-mediated spermagglutination is responsible for infertilityin some couples and fertilization in vitro can also be impairedby these antibodies. Having previously demonstrated the possibilityof enzymatic disagglutination in such situations, the functionalpotential of disagglutinated spermatozoa has now been assessed.Chymotrypsin (500 U/ml) and papain (50 U/ml) resulted in impairmentof oocyte penetration in the zona-free hamster egg penetrationtest. Trypsin (500 U/ml), while having no effect on egg penetrationof normal spermatozoa, significantly improved oocyte penetrationof spermatozoa which had been previously incubated with spermagglutinatingantibody-positive sera. Sperm —mucus interaction was notimproved, however, by trypsin treatment of agglutinated spermatozoa.This technique may be of value in conjunction with in-vitrofertilization in situations where spermagglutination exists,and also possibly with intra-uterine insemination if improvedfertilizing ability can be confirmed in vitro.     

Association of early {beta}-human chorionic gonadotrophin values with pregnancy wastage and multiple implantation in a donor oocyte programme

An early marker predictive of a viable pregnancy would easethe anxiety associated with positive pregnancy tests after theuse of donor oocytes. We examined the predictive value of anearly serum quantitative human chorionic gonadotrophin (Q-HCG)concentration on pregnancy outcome following oocyte donation.Embryo transfers after oocyte donation resulting in a positiveserum -HCG were examined beginning 9 days after embryo transferfrom those samples assayed in our laboratory (n = 77). Q-HCGconcentrations were measured in our laboratory by an immunoradiometricassay utilizing the first International Reference Preparation.Implantations were defined as the number of gestational sacsvisualized by transvaginal ultrasound 21 days after embryo transfer.Biochemical pregnancies were those with transient elevationsin -HCG concentration but without implantation sites. Spontaneousabortions were characterized by an implantation site with theeventual arrest of development. Ongoing/delivered pregnanciesdeveloped appropriately and proceeded beyond the first trimester.Day 9 Q-HCG concentrations did not differentiate between biochemicalpregnancies/spontaneous abortions and ongoing/delivered pregnancies,although mean ± SD concentrations for biochemical pregnancieswere significantly lower than those for the other groups (P< 0.0001): biochemical pregnancies, n = 18, 5.8 ±8.9 mlU/ml, range 0–35; spontaneous abortions, n = 2,46.0 ± 10.0 mlU/ml, range 39–53; ongoing/deliveredpregnancies, n = 57, 41.5 ± 35.4 mlU/ml, range 0–214.In addition, day 9 Q-HCG concentrations did not differentiatebetween multiple implantations, although the implantation offour sacs had a significantly higher mean Q-HCG concentrationcompared with the implantation of fewer sacs (P > 0.0001):one sac, n = 22, 32.2 ± 21.5 mlU/ml, range 3–78;two sacs, n = 25, 35.8 ± 21.3, range 0–81; threesacs, n = 7, 47.1 ± 37.1 mlU/ml, range 22–126;four sacs, n = 4, 122.3 ± 62.4 mlU/ml, range 76–214.The positive predictive value of a Q-HCG >10 mlU/ml was 0.91(sensitivity 91%, specificity 75%). These initial data suggestthat early day 9 serum Q-HCG determinations do not accuratelyidentify viable pregnancies or multiple implantations. Evenan early negative pregnancy test should be repeated becauseit can be associated with a normal pregnancy.     

Abnormal chromosomal arrangements in human oocytes

Ninety-one human oocytes, lacking signs of fertilization 50h after insemination in vitro, were investigated cytogeneticallyto assess the frequency and type of chromosomal abnormalities.Chromosome spreading permitted adequate karyotyping in 55 oocytes.Non-determined numerical aberrations occurred with the followingfrequencies: hypohaploidy, 10.9% (6/55), hyperhapJoidy, 14.5%(8/55) and hyperdiploidy, 3.6% (2/55). Total aneuploidy occurredwith a frequency of 29.1% and was observed in oocytes from 30patients. No correlation was found between specific chromosomalaberrations and type of infertility, stimulation treatment orgonadotrophin levels. On the other hand, the frequency of aneuploidywas significantly higher (P < 0.05) in patients >35 yearsof age. Two chromosomal complements (3.6%) had structural rearrangements;one oocyte had both structural and numerical chromosomal abnormalitiesand the other had differently condensed regions on the longarms of three chromosomes from group C. The overall frequencyof chromosomal aberrations was 32.7%. Only two samples containedan additional set of polar body chromosomes. Thirteen oocytespresented sperm chromosomes in an arrested stage of prematurechromosome condensation of the G₁, phase and four oocytes showedasynchronous condensation of pronuclear chromosomes. Finally,it was concluded that the high proportion of chromosomal aberrationsobserved in human oocytes may contribute significantly to abnormalembryonic development in vitro.     

Endocrinology: Growth hormone, oestradiol, progesterone and testosterone concentrations in follicular fluid after ovarian stimulation with various regimes for assisted reproduction

Follicular fluid samples and oocytes were obtained from 75 women(87 cycles), who participated in an assisted conception programme.Determinations of the concentration of oestradiol, progesterone,testosterone and growth hormone were performed in all follicularfluid samples. Patients were stimulated with the following regimes:group A (24 cycles, 94 samples), human menopausal gonadotrophin(HMG) (three ampoules/day) and human chorionic gonadotrophin(HCG); group B (23 cycles, 53 samples), HMG/HCG with prednisolone(7.5 mg/day) after cycle programming with oral contraceptives;group C (40 cycles, 60 samples), buserelin with HMG/HCG. Oestradiolconcentrations (mean ± SEM) were significantly higher(P < 0.05) in group A (320.1 ± 27.3 ng/ ml) and thoseof growth hormone in both groups A and C (3.8 ± 0.2 and3.2 ± 0.15 ng/ml, respectively), as compared to the othergroups, whereas progesterone and testosterone concentrationswere similar in all groups. The mean concentrations of oestradiol,progesterone, testosterone and growth hormone were significantlyhigher (P < 0.01) in follicular fluid with oocytes of intermediatematurity than with mature oocytes (382.5 ng/ml, 7847.5 ng/ml,1704.5 ng/dl and 3.7 ng/ml versus 217.8 ng/ml, 5488.4 ng/ml,1313.6 ng/dl and 2.7 ng/ml, respectively). On the other hand,only oestradiol concentrations were significantly higher infollicular fluid of fertilized compared to non-fertilized oocytes.Concentrations of the other hormones analysed, except growthhormone, were similar in follicular fluid from pregnant andnon-pregnant women after assisted reproduction. Growth hormone,on the other hand, was significantly lower (P < 0.05) infollicular fluid from pregnant compared to non-pregnant women(2.8 versus 3.5 ng/ml). It is concluded that intermediate maturityoocytes and oocytes which will be subsequently fertilized arefound in follicles with higher follicular fluid concentrationsof growth hormone and steroids. Moreover, oocytes leading topregnancy after in-vitro fertilization and embryo transfer arederived from follicles with lower growth hormone concentrationsin follicular fluid.     

Fertilization and early embryology: The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection

Implementation of intracytoplasmic sperm injection (ICSI) inhuman in-vitro fertilization (IVF) has highlighted the needfor information about the risk of nuclear spindle damage causedby this procedure. For this purpose we studied the final productsof oocyte meiosis at the first cleavage division of multipronuclearzygotes arising after ICSI, and compared the results with abnormallyfertilized oocytes after conventional in-vitro insemination.Of 37 successfully analysed tripronuclear zygotes, 18 had threeindividual metaphases. Abnormal complements of 11 zygotes inthis group indicated that non-disjunction occurred predominantlyat the second meiotic division of the oocytes. Nine of the 37tripronuclear zygotes exhibited two individual metaphases. Sevenwere abnormal and there were some indications that non-disjunctiontook place during oocyte meiosis. Of the 37 tripronuclear zygotes,10 had a single metaphase and three showed an aneuploid numberof chromosomes. The overall rate of aneuploidy among tripronuclearmicroinjected zygotes was 56.7%. In addition, seven zygoteswith more than three pronuclei arising after ICSI displayedseverely depleted chromosome complements. The incidence of non-disjunctionin oocytes fertilized by conventional in-vitro inseminationwas significantly lower (20.0%, P < 0.01), since only fourzygotes had an aneuploid number of chromosomes. Our findingssuggest that ICSI might interfere with regular chromosome segregationat the second meiotic division of the oocytes.     

Fertilization and early embryology: Timing of pronuclear development and first cleavages in human embryos after subzonal insemination: influence of sperm phenotype

The sequential transformations of human sperm nuclei in humaneggs after subzonal insemination (SUZI; n = 104) and the influenceof sperm defects on this timing were studied. This chronologywas compared to that of two control series of zygotes obtainedafter SUZI with normal spermatozoa (n = 35) and after in-vitrofertilization (IVF) with normal donor spermatozoa (D-IVF; n= 220). Pronuclear formation took place between 4.5 and 10.5h post-SUZI for 92.8% of the zygotes. They remained visiblefor 13 h and began to disappear 18.5 h post-SUZI. The time-spanbetween pronuclear disappearance and cleavage was 3 h. Zygotesobtained after D-IVF had a similar rate of pronuclear disappearancebut 4 h later. The second cell cycle was more rapid for zygotesobtained by D-IVF than by SUZI, but the developmental rate ofzygotes obtained by SUZI varied according to sperm phenotypes.For patients with previous unexplained IVF failures (controlgroup with normal spermatozoa), the developmental rate was lower,suggesting the influence of oocyte quality. In conclusion, theend of the first cell cycle of zygotes obtained by inseminationunder the zona pellucida appears 4 h earlier compared to zygotesobtained after insemination outside the zona pellucida.