Planned oocyte cryopreservation (Planned OC): systematic review and meta-analysis of cost-efficiency and patients' perspective.

BACKGROUND: Advances in vitrification techniques have enabled planned oocyte cryopreservation ('Planned OC'). OBJECTIVES: To explore the cost-efficiency and utilisation of planned OC, as well as patients' perspectives on the process. SEARCH STRATEGY: A systematic search in PubMed/MEDLINE, Embase, Cochrane Database and PsychINFO, for all relevant studies published between January 2007 and December 2019. SELECTION CRITERIA: The protocol followed PRISMA guidelines in PECO format, and was registered with PROSPERO. DATA COLLECTION AND ANALYSIS: Two independent reviewers evaluated all manuscripts for inclusion eligibility. Authors were contacted for missing data. Included studies were assessed for risk of bias and for heterogeneity. Weighted effects were measured and plotted. MAIN RESULTS: The search yielded 12 545 records, of which 43 were included. Planned OC is cost-efficient at 35, assuming 60% utilisation; and at 37 assuming utilising donor sperm when necessary. At 38 it is cost-efficient to defer planned OC in favour of undergoing 2 IVF cycles. Currently, utilisation of banked-oocytes within 22-58 months, is up to 15%. Nine percent of warmed banked oocytes result in life births. Online resources and treating physicians are equally important sources of information regarding planned OC. Most patients think planned OC is ideal before age 35 and are not fully aware of what the process entails and tend to overestimate the success rates. The main barrier to wider endorsement of planned OC is being wary of potential health implications or of limited success. CONCLUSION: Planned OC is an adequate method for preserving fertility. However, knowledge gaps result in under-utilisation leading to reduced cost-efficiency. TWEETABLE ABSTRACT: Identifying facilitators and barriers for wider adoption of banking oocytes can enhance the cost-efficiency of this modality.

Potential use of stem cells for fertility preservation.

BACKGROUND: Infertility and gonadal dysfunction can result from gonadotoxic therapies, environmental exposures, aging, or genetic conditions. In men, non-obstructive azoospermia (NOA) results from defects in the spermatogenic process that can be attributed to spermatogonial stem cells (SSC) or their niche, or both. While assisted reproductive technologies and sperm banking can enable fertility preservation (FP) in men of reproductive age who are at risk for infertility, FP for pre-pubertal patients remains experimental. Therapeutic options for NOA are limited. The rapid advance of stem cell research and of gene editing technologies could enable new FP options for these patients. Induced pluripotent stem cells (iPSC), SSC, and testicular niche cells, as well as mesenchymal stromal cells (aka medicinal signaling cells, MSCs), have been investigated for their potential use in male FP strategies. OBJECTIVE: Here, we review the benefits and challenges for three types of stem cell-based approaches under investigation for male FP, focusing on the role that promising sources of MSC derived from human umbilical cord, specifically human umbilical cord perivascular cells (HUCPVC), could fulfill. These approaches are as follows: 1. isolation and ex vivo expansion of autologous SSC for in vivo transplantation or in vitro spermatogenesis; 2. in vitro differentiation toward germ cell and testicular somatic cell lineages using autologous SSC, or stem cells such iPSC or MSC; and 3. protection or regeneration of the spermatogenic niche after gonadotoxic insults in vivo. CONCLUSION: Our studies suggest that HUCPVC are promising sources of cells that could be utilized in multiple aspects of male FP strategies.

A combination of single nucleotide polymorphisms in the 3'untranslated region of HLA-G is associated with preeclampsia.

Reduced expression of human leukocyte antigen-G (HLA-G) has been linked to onset of preeclampsia. Associations have also been reported between preeclampsia and single nucleotide polymorphisms (SNP) in the 3'-untranslated region (UTR) of the HLA-G gene. However, there are conflicting results between studies. This studied examined whether a SNP, by itself or in combination with other SNPs, in the 3'UTR of the HLA-G gene is associated with an increased risk of preeclampsia. Placenta samples were obtained from 47 preeclamptic and 68 control cases. DNA was extracted, and the 3'UTR was sequenced and analyzed for nine polymorphisms using different genetic models of inheritance. Four of these polymorphisms have never been analyzed for an association with preeclampsia. Disputing existing reports, preeclamptic cases were suggestively associated with a G/G-genotype at SNP +3187 (p<0.05). Several SNP combinations were more prevalent in preeclampsia cases. Following corrections for multiple testing, one SNP combination (+3027C/C and +3187G/G) was significantly more prevalent in preeclampsia cases using co-dominant, additive, and dominant models (p<0.001). Taken together with the current literature, the data suggests that HLA-G 3'UTR SNP-pair associations, and not individual SNPs, could be useful in a predictive test for the susceptibility to preeclampsia.

Neonatal outcomes among singleton births after blastocyst versus cleavage stage embryo transfer: a systematic review and meta-analysis.

BACKGROUND: Several studies have evaluated outcomes of singleton pregnancies after blastocyst versus cleavage stage embryo transfer. Higher incidences of preterm birth (PTB), very preterm birth (VPTB), low birthweight (LBW) and congenital malformations were identified in a few of them. The objective of our study was to systematically review and meta-analyze pregnancy and neonatal outcomes among singleton births following blastocyst versus cleavage stage embryo transfer. METHODS EMBASE, MEDLINE, EBM Reviews and bibliographies of included studies were searched from their inception until March 2013. Observational studies or clinical trials comparing blastocyst with cleavage stage embryo transfer and reporting on outcomes of PTB (<37 weeks), VPTB (<32 weeks), LBW (<2500 g), very low birthweight (VLBW) (<1500 g) and/or congenital anomalies in singleton neonates were included. Data on the outcomes were extracted by two reviewers. Statistical heterogeneity among studies was evaluated by calculating I(2) values and χ(2) statistics. Meta-analyses were conducted to estimate the pooled unadjusted odds ratio (OR) and the adjusted OR (AOR) with a 95% confidence interval (CI) using the random effect model. RESULTS Six observational studies, of low to moderate risk of bias, were included in this review. There were significantly higher odds of PTB (four studies, 54 792 cleavage stage and 20 724 blastocyst stage births; AOR 1.32, 95% CI 1.19-1.46) and congenital anomalies (two studies, 22 068 cleavage stage and 4517 blastocyst stage births; AOR 1.29, 95% CI 1.03-1.62) among births after blastocyst transfer compared with cleavage stage transfer. There was no difference in the adjusted odds of VPTB (four studies, 54 792 cleavage stage and 20 724 blastocyst stage births; AOR 1.18, 95% CI 0.93-1.49), LBW (four studies, 54 109 cleavage stage and 20 392 blastocyst stage births; AOR 1.06, 95% CI 0.99-1.15) or VLBW (three studies, 22 088 cleavage stage and 5772 blastocyst stage births; AOR 1.01, 95% CI 0.73-1.38). CONCLUSIONS Risk of PTB in IVF singleton pregnancies is significantly higher following blastocyst transfer compared with cleavage stage transfer. Risk of congenital anomalies may also be higher but further studies are needed to confirm this finding and to identify reasons for such outcomes.

Increased risk of preterm birth in singleton pregnancies after blastocyst versus Day 3 embryo transfer: Canadian ART Register (CARTR) analysis.

STUDY QUESTION: Are the fetal outcomes of singleton pregnancies that result from cleavage stage embryo transfer (ET) different from the outcomes from Day 5/6 blastocyst stage ET? SUMMARY ANSWER: There was a significantly higher risk of preterm birth (<37 weeks) in singletons after extended embryo culture (Day 5/6) compared with cleavage stage (Day 3) transfer. WHAT IS KNOWN ALREADY: Two recent studies, from Sweden and the USA, reported an increased risk of preterm birth in singleton pregnancies after Day 5/6 ET compared with Day 3 ET. The US study also showed increased early preterm births and the Swedish study showed increased fetal malformations in this group. STUDY DESIGN, SIZE AND DURATION: A retrospective cohort study was performed. Data were collected from the Canadian ART Register database for all singleton births after fresh IVF/ICSI ET cycles (2001-2009). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 12 712 singleton births were included. Of these, 9506 resulted from a Day 3 ET and 3206 resulted from a blastocyst (Day 5/6) ET. MAIN RESULTS AND THE ROLE OF CHANCE: Preterm birth rate <37 weeks (unadjusted by potential confounding factors) was higher with Day 5/6 versus Day 3 transfers (17.2 versus 14.1%, P < 0.001). Using logistic regression analysis to adjust for confounding factors, preterm birth rate <37 weeks was the only outcome significantly increased after Day 5/6 compared with Day 3 transfer (odds ratio 1.32, 95% confidence interval 1.17-1.49). The following confounding factors were adjusted for: year of treatment (2001-2009), maternal age (continuous), parity (0 versus ≥1 birth), diagnosis category, number of oocytes retrieved [≤20 versus >20 (high responder group)], insemination method (IVF versus ICSI), number of embryos transferred (1, 2 or ≥3) and the presence of a vanishing twin (≥1 fetal heart on the initial ultrasonographic examination). LIMITATIONS, REASONS FOR CAUTION: Post-natal follow-up studies will be required to determine if this difference we observed translates into adverse long-term effects on these offspring. The rate of early preterm births (<32 weeks) was higher in Day 5/6 versus Day 3, but the low number of cases in this category did not have the power to show a difference (3.0 versus 2.7%, P = 0.34). WIDER IMPLICATIONS OF THE FINDINGS: We found a significantly higher risk of preterm birth (<37 weeks) in singletons after extended embryo culture (Day 5/6) compared with cleavage stage (Day 3) transfer, even when adjusting for confounding factors. Our findings are in agreement with the previous two studies; however, we did not show a difference in the very preterm deliveries (unlike the US study) or in fetal malformations (as in the Swedish study). We hypothesize that there may be a deleterious effect of prolonged in vitro embryo culture on subsequent placentation. Longer term follow-up studies will be required to determine if prolonged in vitro culture to the blastocyst stage has an adverse effect on the long-term health of offspring when compared with shorter cleavage stage culture. STUDY FUNDING/COMPETING INTEREST(S): None.

HLA-G has a concentration-dependent effect on the generation of an allo-CTL response.

Human leucocyte antigen (HLA) -G is expressed on trophoblast cells during pregnancy, suggesting a role in protection of the semiallogeneic fetus. Published data suggest that HLA-G protects a cell against natural killer cell lysis. It has been hypothesized that HLA-G may also protect the fetus by preventing allo-cytotoxic T lymphocyte (CTL) responses. To test this hypothesis, we assayed the effects of various concentrations of purified HLA-G on CTL response in a mixed lymphocyte culture (MLC) system. We found that concentrations > or =0.1 microg/ml of HLA-G suppressed the allo-CTL response by 30-100% over the control, but, paradoxically, concentrations of 0.01-0.05 microg/ml of HLA-G augmented the allo-CTL response by 25-50% over the control. Concentrations < or = 0.001 microg/ml HLA-G had no effect. Addition of HLA-G to preprimed allo-CTL effector cells did not affect their killing ability. Allo-CTL suppressive doses of HLA-G induced a T helper type 2 (Th2) cytokine response, whereas allo-CTL-enhancing doses of HLA-G induced a Th1-type cytokine response. HLA-G purified from first-trimester placenta does not affect allo-proliferative responses nor does it alter the percentage of CD4+ or CD8+ T cells in MLCs. These findings support a potential role for HLA-G-mediated suppression of allo-CTL formation in normal pregnancies. In addition, the effects observed at lower concentrations of HLA-G may have interesting implications for the condition of pre-eclampsia in which concentrations of this HLA class I molecule are reduced.

Embryonic human leukocyte antigen-G expression: possible implications for human preimplantation development.

OBJECTIVE: To investigate further the association between human leukocyte antigen G (HLA-G) expression in human embryos and other factors known to influence IVF pregnancy outcome. SETTING: A university-based tertiary referral center (The Toronto Hospital). INTERVENTIONS: Nontransferred embryos at the two- to four-cell stage were obtained from patients undergoing IVF and were cultured in Ham's F-10 medium supplemented with 10% human sera or cocultured with ovarian cancer cells in the same medium. Embryos that reached blastocyst stage (n = 148) were analyzed by reverse transcriptase-polymerase chain reaction for HLA-G and beta 2 microglobulin (beta 2m) expression. Statistical analysis was performed to identify possible factors associated with variability of expression. RESULTS: Approximately 40% of studied blastocysts had detectable expression of both HLA-G and beta 2m messenger RNA. In 46% of blastocysts, beta 2m alone was observed. Interestingly, sibling embryos from patients that became pregnant were significantly more likely to express HLA-G than embryos from patients that did not conceive as a result of their IVF cycles. No association was found between HLA-G expression and culture conditions, patients age, or infertility diagnosis. CONCLUSION: The population of embryos obtained through IVF is heterogeneous in expression of HLA-G and beta 2m, which may reflect overall health of the embryos. Blastocysts showing positive HLA-G expression may have increased viability and implantation potential, although the underlying mechanisms remain to be elucidated.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Interleukin-1 beta regulates human cytotrophoblast metalloproteinase activity and invasion in vitro.

During early human pregnancy, fetal cytotrophoblasts rapidly invade the uterus. This process has many similarities to tumor invasion, except that the extent and the timing of cytotrophoblast invasion are carefully regulated. Therefore, this system is particularly useful for studying mechanisms that regulate invasive processes. Previously, we showed that production and activation of the 92-kDa type IV collagenase (matrix metalloproteinase(MMP)-9) is necessary for cytotrophoblast invasion in vitro. In other systems, interleukin (IL)-1 beta is an important regulator of matrix-degrading metalloproteinases. Therefore, we investigated trophoblast production of IL-1 beta and its receptors, as well as the effects of this cytokine on cytotrophoblast metalloproteinase activity and invasion. The results showed that release of IL-1 beta parallels the invasive potential of the cytotrophoblasts; the highest levels are produced by first trimester cells and the lowest levels by term cells. Immunoprecipitation showed that cytotrophoblasts express the 80-kDa type I IL-1 receptor, suggesting that autocrine effects are possible. IL-1 beta stimulated trophoblast MMP-9 secretion (by a mechanism that required nascent mRNA and protein synthesis) as well as metalloproteinase activity and invasion of Matrigel. Increasing (by lipopolysaccharide treatment) or decreasing (by glucocorticoid treatment) IL-1 beta production had parallel effects on MMP-9 secretion, metalloproteinase activity, and invasion. Because IL-1 beta and corticosteroids are present in high concentrations at the maternal-fetal interface, normal trophoblast invasion may be regulated, in part, by their opposing actions. In contrast, stimulation of cytotrophoblast IL-1 beta secretion by lipopolysaccharide may play a role in the sequela of infected fetal membranes.

Localization and regulation of the activin-A dimer in human placental cells.

Subunits of activin and inhibin and their mRNAs are present in human placental and decidual cells. However, evidence for the presence of intact activin dimers in the human placenta and their regulation has been lacking. Using a monoclonal antibody raised against the human activin-A dimer, we examined the cellular localization of immunoreactive activin-A dimer in human placentas of different gestational ages (8-41 weeks). In addition, we determined the effects of culture and various potential regulators on the cellular accumulation of immunoreactive activin-A dimer in trophoblast cells from human first trimester placentas. Activin-A dimer was found in both cyto- and syncytiotrophoblast cells of all gestational ages studied. Immunoreactive activin-A also was detected in placental Hofbauer cells in first and second trimester placentas as well as in cells of the placental membranes. Exposure of these cells to cAMP, GnRH, activin, inhibin, transforming growth factor-beta, dexamethasone, and interleukin-1 did not significantly change the intensity of immunostaining for activin-A dimer. These results together with previous data suggest that placental cells are a source of activin-A and that activin-A may be a paracrine and/or endocrine regulator of feto-maternal interactions during pregnancy.

92-kD type IV collagenase mediates invasion of human cytotrophoblasts.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.

Weights of fetuses with autosomal trisomies at termination of pregnancy: an investigation of the etiologic factors of low serum alpha-fetoprotein values.

In order to determine whether the low values of maternal serum alpha-fetoprotein observed with autosomal trisomies are associated with smaller fetal weights, 50 fetuses with Down syndrome (trisomy 21), 10 with trisomy 18, and 65 normal control fetuses, all aborted in the second trimester of pregnancy, were compared. The mean multiple of the median maternal serum alpha-fetoprotein was found to be 0.79 +/- 0.61 for fetuses with Down syndrome and 0.50 +/- 0.26 for those with trisomy 18, both results being significantly lower than results from the control fetuses (0.97 +/- 0.86). No significant difference in the weight distribution between fetuses with Down syndrome and control fetuses, corrected for gestational age, was found. By contrast, fetuses with trisomy 18 had a significantly lower weight distribution compared with that of the control fetuses (p less than 0.001). A linear relationship was found in normal fetuses between maternal serum alpha-fetoprotein values and fetal weight at a given gestational age. Fetal weight does not seem to account for the lower maternal serum alpha-fetoprotein levels seen in fetuses with Down syndrome but may partially account for the lower levels seen in fetuses with trisomy 18.

The influence of prolonged pregnancy on infant development at one and two years of age: a prospective controlled study.

Isolated reports of developmental disturbances following prolonged pregnancy led us to compare, prospectively, at 1 and 2 years of age, infants born after normal term gestations with those born after prolonged pregnancies (exceeding 294 days). The infants were subgrouped according to their physical condition at birth, that is, normal or dysmature (mild or advanced dysmaturity). Infant assessments included: (1) height and weight, (2) hospitalizations, and (3) mental development by the Griffiths Mental Development Scales. Follow-up testing was obtained on 130 term control infants and 89 infants of prolonged pregnancies at 1 year of age and 111 term control infants and 76 infants of prolonged pregnancies at 2 years of age. At 1 and 2 years the general intelligence quotient, physical milestones, and intercurrent illnesses for normal infants and those of prolonged pregnancies were not significantly different.

Microsurgical reversal of sterilization: a six-year study.

A prospective study of 113 personal consecutive microsurgical reversals of female sterilization during the 6-year period from 1979 to 1984 was carried out to determine factors affecting the pregnancy rate. The sterilizations were performed by laparoscopic unipolar coagulation in 54% of the patients, by the Pomeroy technique in 28%, by fimbriectomy in 8%, by the Irving operation in 5%, and by clips or rings in 4%. In the group with no minimum follow-up period, 50% had intrauterine pregnancies and 5% had ectopic gestations. Eighty-nine patients had at least 12 months of follow-up after reversal surgery. This group is studied in detail. Factors affecting the pregnancy rate were length of tube, type of sterilization performed, anastomotic site, and availability of both tubes for reconstruction. Age, parity, and interval from sterilization to reversal surgery did not affect the pregnancy rate. Fifty percent of the intrauterine pregnancies were conceived within 6 months of reversal surgery.

Genetic amniocentesis in seventy twin pregnancies.

Genetic amniocenteses were performed in 70 twin pregnancies over an 11-year period. Both sacs were successfully sampled in 49 of 62 patients (79%). The success rate was decreased (68%) with two placentas (anterior and posterior) and was improved with gestational age greater than or equal to 17 weeks (88%) and with ultrasound visualization of the septum (86%). Of three spontaneous abortions, two were attributed to amniocentesis (chorioamnionitis). When twin pregnancy is diagnosed in a patient with an indication for genetic amniocentesis, a careful reevaluation and discussion of risk factors with the couple are recommended.

Spontaneous reverse haemolytic plaque formation. II. The role of T cells and monocytes in regulating immunoglobulin secretion by human peripheral blood B cells.

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94%; this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10-1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1, this significantly reduced or abolished spontaneous IgG plaques, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for monocytes, OKM1 and FMC17, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors.

Rapid replicate reverse haemolytic plaque assay directly in microwells used for tissue culture.

The reverse haemolytic plaque assay was adapted as a micromethod using microwells of flat-bottomed microtitre trays. To microwells containing the lymphocytes under test were added protein A-coupled ox erythrocytes, developing antisera directed against any class of immunoglobulin, and guinea pig complement absorbed with protein A-coupled erythrocytes. Plaques were scored by counting with a stereoscan microscope. The method is applicable both to spontaneous and mitogen-induced plaque formation and, as a further development of the technique to differentiated B cells in pokeweed mitogen-driven cultures, plaques were enumerated directly in the same wells of the microtitre tray as were employed for culture of the B cells. The method allows for several hundred plaque assays at one sitting.