Correlations of the expression of γδ T cells and their co-stimulatory molecules TIGIT, PD-1, ICOS and BTLA with PR and PIBF in the peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion.

Semi-allogeneic embryos are not rejected by the maternal immune system due to maternal-fetal immune tolerance. Progesterone (P) receptor (PR)-expressing γδ T cells are present in healthy pregnant women. In the presence of P, these cells secrete an immunomodulatory protein called progesterone-induced blocking factor (PIBF), which can facilitate immune escape and is important in preventing embryonic rejection. This work investigated the correlations of the expression of γδ T cells and their co-stimulatory molecules T cell immunoglobulin and ITIM domain (TIGIT), programmed cell death 1 (PD-1), inducible co-stimulator (ICOS) and B and T lymphocyte attenuator (BTLA) with progesterone receptor (PR) and progesterone-induced blocking factor (PIBF) in peripheral blood and decidual tissue in women with unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women. We confirmed that γδ T cell proportions and PIBF expression in the peripheral blood and decidua of URSA women decreased significantly, while PR expression in decidua decreased. However, TIGIT, PD-1, ICOS and BTLA expression in γδ T cells in peripheral blood did not change, while TIGIT and PD-1 expression in γδ T cells in decidua increased significantly. Under the action of PHA-P (10 µg/ml), co-blocking of TIGIT (15 µg/ml) and PD-1 (10 µg/ml) antibodies further induced γδ T cell proliferation, but PIBF levels in the culture medium supernatant did not change. At 10-10 M P, γδ T cells proliferated significantly, and PIBF concentrations in the culture medium supernatant increased. γδ T cells co-cultured with P, TIGIT and PD-1 blocking antibodies showed the most significant proliferation, and PIBF concentrations in the culture medium supernatant were the highest. These results confirm that P is necessary for PIBF production. The TIGIT and PD-1 pathways participate in γδ T cell proliferation and activation and PIBF expression and play important roles in maintaining pregnancy.

In-depth blood proteome profiling analysis revealed distinct functional characteristics of plasma proteins between severe and non-severe COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over forty million patients worldwide. Although most coronavirus disease 2019 (COVID-19) patients have a good prognosis, some develop severe illness. Markers that define disease severity or predict clinical outcome need to be urgently developed as the mortality rate in critical cases is approximately 61.5%. In the present study, we performed in-depth proteome profiling of undepleted plasma from eight COVID-19 patients. Quantitative proteomic analysis using the BoxCar method revealed that 91 out of 1222 quantified proteins were differentially expressed depending on the severity of COVID-19. Importantly, we found 76 proteins, previously not reported, which could be novel prognostic biomarker candidates. Our plasma proteome signatures captured the host response to SARS-CoV-2 infection, thereby highlighting the role of neutrophil activation, complement activation, platelet function, and T cell suppression as well as proinflammatory factors upstream and downstream of interleukin-6, interleukin-1B, and tumor necrosis factor. Consequently, this study supports the development of blood biomarkers and potential therapeutic targets to aid clinical decision-making and subsequently improve prognosis of COVID-19.

Progesterone induced blocking factor (PIBF) taken in early pregnancy predicts the pregnancy outcome in women undergoing in vitro fertilization procedure.

Earlier data suggest a relationship between PIBF concentrations and the outcome of pregnancy. The aim of the study was to compare serum and urine concentrations of PIBF in women with successful pregnancy after IVF with those of women without pregnancy after IVF procedure, and to evaluate the potential relation between PIBF and the outcome of pregnancy. Urine and serum were collected from 120 women, undergoing IVF. 87.5% of patients had primary infertility. 69.2% faced female causes of infertility: 10.8% tubal cause, 11.7% ovulation disorder, and 46.7% other causes of infertility. 30.8% of patients had male factor of infertility. Among non-pregnant women (42) mean concentrations of PIBF in urine and serum were significantly lower (15.8 ng/mL; 148.4 ng/mL) than in women with positive beta HCG value (78) (19.1 ng/mL; 225.9 ng/mL). In 49 patients pregnancy terminated with a term delivery, in 10 patients with pretem delivery, while in 19 patients the pregnancy terminated with a miscarriage. PIBF concentrations in urine (13.9 ± 2.8 ng/mL) and serum (124.6 ± 46.7 ng/mL) samples of women with miscarriage were significantly lower of those with preterm delivery (180.6 ± 54.4 ng/mL; 18.1 ± 4.4 ng/mL) and of those with term delivery (20.4 ± 8.5 ng/mL; 208.7 ± 114.3 ng/mL). Successful pregnancy after IVF procedure is predictable by measuring of urine and serum PIBF concentrations and could be important for predicting of early implantation and pregnancy outcome after IVF procedure and maybe to protect the risk pregnancy.

Characterisation of serum progesterone and progesterone-induced blocking factor (PIBF) levels across trimesters in healthy pregnant women.

Progesterone-induced blocking factor (PIBF), which plays an important role in maintaining healthy pregnancies, has shown great promise as a prognostic biomarker for threatened miscarriage. To better characterise the physiological trends of progesterone and PIBF, we analysed serum progesterone and PIBF concentrations in healthy non-pregnant and pregnant women across trimesters. We saw increasing concentrations of progesterone and PIBF in pregnant women with advancing trimesters. The serum progesterone and PIBF percentiles across gestational age in healthy pregnancies can be used as a guide for the formulation of reference ranges. We also demonstrated a significant positive correlation between progesterone and PIBF levels. This study demonstrates increasing progesterone and PIBF concentrations in later trimesters and underscores the importance of progesterone and PIBF in healthy pregnancies. Characterisation of progesterone and PIBF across gestational age in healthy pregnant women may help to prognosticate pregnancy viability and support further research into the importance of progesterone and PIBF in the maintenance of healthy pregnancies.

Serum levels of the immunomodulatory protein, the progesterone induced blocking factor (PIBF) which is found in high levels during pregnancy is not higher in women with progesterone (P) receptor (R) positive vs. negative breast cancer.

PURPOSE: To determine if serum levels of the immunomodulatory protein, the progesterone induced blocking factor (PIBF), which is present in high levels during normal pregnancy, is present in higher levels in women with breast cancer positive for progesterone receptors. The study would also determine whether the presence or absence of the estrogen receptor in any way modifies PIBF expression. MATERIALS AND METHODS: PIBF using a research ELISA was evaluated in the follicular phase in 21 women with receptor status as follows: seven with estrogen receptor (ER)+ and progesterone receptor (PR)+, seven with ER- and PR+, and seven with ER+ and PR. RESULTS: The results showed no differences in serum PIBF in the three groups. The serum PIBF levels were no different than historical controls in the follicular phase. CONCLUSIONS: Measurement of serum PIBF does not seem to be an important marker to use to either detect women with breast cancer or to help determine tumor virulence or potential specific therapies. If PIBF plays a role in helping cancer cells to escape immune surveillance, it seems that the intracytoplasmic PIBF would be the form most likely operative.

Lower Urinary and Serum Progesterone-Induced Blocking Factor in Women with Preterm Birth.

The aim of the study was to compare urine and serum concentrations of PIBF at 24-28 gestational weeks in women with preterm birth, with those of women who delivered at term and to evaluate the impact of PIBF on the outcome of pregnancy. Case-control study was performed in period from 1.6.2010-31.7.2013. Biological samples (urine and serum) were collected from 126 pregnant women. All biological samples were obtained at 24-28 gestation weeks. We measured PIBF concentration and compared women who delivered preterm and those who delivered at term. Thirteen of 126 pregnant women (10.3%) who were included in the study delivered preterm. Among women that actually delivered preterm, median concentrations of PIBF were significantly lower (12.3ng/ml; 101.3ng/ml) than in women who delivered at term (77.0ng/ml; 412.7ng/ml). The serum and urine 24-28 gestational weeks PIBF in those who delivered preterm were generally low from 24 to 37 gestational weeks, while the serum and urine PIBF concentration reached a peak in those delivering between 37-38 gestational weeks, even significantly different from those delivering at 39 to 40 and after 40 gestational weeks. Preterm birth may be predictable at 24-28 gestational week by lower than normal pregnancy PIBF values and measurement of PIBF concentration in biological fluids at that time may be of importance in clinical practice.

The progesterone receptor antagonist mifepristone does not lower serum progesterone induced blocking factor (PIBF) in the presence of progesterone.

PURPOSE: To determine if mifepristone can lower serum levels of a progesterone (P) induced immunomodulatory protein believed to be needed for the fetus to escape immune surveillance. MATERIALS AND METHODS: A female volunteer had her serum P induced blocking factor (PIBF) increased by ingestion of oral micronized P. While remaining on P mifepristone, 200 mg/day was given for six days when another serum PIBF level was obtained. RESULTS: The serum PIBF was 273 ng/ml after five days of oral micronized P. It increased further to 737 ng/ml despite taking six days of 200 mg mifepristone. CONCLUSIONS: The mechanism for inducing abortion by mifepristone does not seem to be related to decreasing serum levels of PIBF. This does not eliminate the possibility that the mechanism involves reducing the intracytoplasmic PIBF levels.

Evidence that exposure to progesterone alone is a sufficient stimulus to cause a precipitous rise in the immunomodulatory protein the progesterone induced blocking factor (PIBF).

PURPOSE: To determine if exposure to progesterone alone is sufficient to increase the production of the immunomodulatory protein known as the progesterone induced blocking factor (PIBF). Also to determine what method of progesterone delivery or form of P best stimulates PIBF secretion. METHODS: Serum samples from patients with infertility and paid volunteers were evaluated for both PIBF and progesterone at various times during the follicular phase and the luteal phase in both natural cycles and cycles involving embryo transfer after endogenous and exogenous progesterone exposure and after various synthetic progestins. PIBF was measured by a non-commercial research ELISA assay. Comparisons were made of serum PIBF before and after exposure to progesterone, 17-hydroxyprogesterone, and oral contraceptives. PIBF was also measured before and after transfer of embryos. RESULTS: Progesterone alone without exposure to the fetal allogeneic stimulus was able to produce a marked increase in serum PIBF. Neither a synthetic progestin (19-nortestosterone derivative) nor 17-hydroxyprogesterone caused an increase in PIBF. Some PIBF is generally detected even in the follicular phase. CONCLUSIONS: A previous concept considered that an allogeneic stimulus, e.g., from the fetal semi-allograft, was necessary to induce de novo progesterone receptors in gamma delta T cells, which, in turn, when exposed to a high concentration of progesterone, would secrete high levels of PIBF. These data show that exposure to an allogeneic stimulus is not needed to cause a marked rise in PIBF, merely progesterone alone is sufficient.

Serum levels of the progesterone induced blocking factor do not precipitously rise in women with gynecologic cancer in contrast to women exposed to progesterone.

PURPOSE: To determine if an immunomodulatory protein (progesterone induced blocking factor [PIBF]) that is progesterone induced and found in higher concentration during pregnancy is similarly found with increased levels in women with gynecologic cancers. MATERIALS AND METHODS: A newly developed enzyme linked immunoabsorbent assay (ELISA) assay was used to measure PIBF in the sera of six women with various gynecologic cancers and compare them to five controls (three with benign tumors and two having gynecologic procedures for non-tumors. RESULTS: The PIBF levels in women with gynecologic cancer did not rise precipitously as historical controls of women or men exposed to progesterone. The two highest PIBF levels of the 11 subjects were in women with gynecologic cancer. CONCLUSIONS: The data suggest that if PIBF helps cancer cells to evade immune surveillance, it probably operates through an intracytoplasmic presence. If an increase in sera PIBF could have been detected in women with gynecologic cancer, then this ELISA test could have been used to detect tumor recurrence. Future studies may concentrate on evaluating intracytoplasmic PIBF to possibly help determine which tumors may respond to progesterone antagonist receptors.

[Expression of progesterone-induced blocking factor in severe preeclampsia and its association with immune tolerance imbalance].

OBJECTIVE: To explore progesterone-induced blocking factor (PIBF) expression in the placenta and blood of patients with severe preeclampsia and its relationship with immune tolerance imbalance. METHODS: Forty-seven patients admitted between January and December, 2012 were enrolled in this study, including 25 patients with early-onset severe preeclampsia (EOPE) and 22 with late-onset severe preeclampsia (LOPE), with 25 women with normal pregnancy serving as control group. The antenatal blood and postpartum placenta were collected for immunohistochemical staining to detect PIBF expression in the placenta and for testing serum PIBF level using ELISA. Flow cytometry was used to detect the percentage of circulating Th1 and Th2 cells and the Th1/Th2 ratio was calculated. RESULTS: PIBF was expressed in decidual cells, syncytiotrophoblasts and partial cytotrophablasts. The serum PIBF levels were 213.58 ± 44.93 ng/ml in EOPE group, 243.00∓61.19 ng/ml in LOPE group and 273.91 ± 48.57 ng/ml in control group. There were significant differences in serum PIBF, blood Th1/Th2 and placenta PIBF-IOD among the 3 groups (P<0.05). EOPE group had significantly lower serum PIBF, lower llacental PIBF quantity (PIBF-IOD) and higher blood Th1/Th2 than the control group (P<0.05). Serum PIBF in women with severe preeclampsia was positively correlated with placenta PIBF-IOD and negatively with blood Th1/Th2 ratio (P<0.05), but a negative correlation between serum PIBF and 24-hour urinary protein was found only in EOPE group (P<0.05). CONCLUSION: The immune tolerance imbalance mediated by PIBF may participate in the pathogenesis of severe preeclampsia. PIBF, the immune suppressor secreted by lymphocytes of pregnancy women, is also a protective factor against severe preeclampsia, which is expected to be a new target in therapy.

17ß-estradiol contributes to the accumulation of myeloid-derived suppressor cells in blood by promoting TNF-α secretion.

Estrogens are strongly implicated in gender differences in immune responses by influencing the development and activation of immune cells. Recent studies have shown that myeloid-derived suppressor cells (MDSCs), derived from CD11b(+)Gr-1(+) myeloid cells under pathological conditions, play vital roles in modulating immune responses. However, it is still unknown the effects of estrogens on MDSCs. In the present study, we investigated the effects and mechanisms of estrogens on regulating the accumulation of MDSCs. It was found that, compared with male patients with systemic lupus erythematosus (SLE), female patients with SLE showed a higher frequency of MDSCs in peripheral blood mononuclear cells and a higher level of tumor necrosis factor α (TNF-α) in serum. Notably, estradiol level in the serum of female patients with SLE was positively correlated with the frequency of MDSCs. Moreover, 17ß-estradiol could promote TNF-α-induced accumulation of MDSCs in vivo by increasing the fundamental frequency of CD11b(+)Gr-1(+) cells. Furthermore, 17ß-estradiol promoted the secretion of TNF-α in vivo, which contributed to the increase of the frequency of CD11b(+)Gr-1(+) cells. In addition, it was also found that female mice showed a higher frequency of CD11b(+)Gr-1(+) cells and a higher TNF-α level in blood than the age-matched male mice. These data indicate that 17ß-estradiol contributes to the accumulation of MDSCs in blood by promoting TNF-α secretion, which increases the fundamental frequency of CD11b(+)Gr-1(+) cells. Our findings provide a new insight into the mechanism of gender difference in the prevalence of inflammation and autoimmune diseases.

How can we better predict the risk of spontaneous miscarriage among women experiencing threatened miscarriage?

This study seeks to establish progesterone and progesterone-induced blocking factor (PIBF) levels as predictors of subsequent completed miscarriage among women presenting with threatened miscarriage between 6 and 10 weeks of gestation. Our secondary objective was to assess the known maternal risk factors, toward development of a parsimonious and clinician-friendly risk assessment model for predicting completed miscarriage. In this article, we present a prospective cohort study of 119 patients presenting with threatened miscarriage from gestation weeks 6 to 10 at a tertiary women's hospital emergency unit in Singapore. Thirty (25.2%) women had a spontaneous miscarriage. Low progesterone and PIBF levels are similarly predictive of subsequent completed miscarriage. Study results (OR, 95% CI) showed that higher levels of progesterone (0.91, 95% CI 0.88-0.94) and PIBF (0.99, 95% CI 0.98-0.99) were associated with lower risk of miscarriage. Low progesterone level was a very strong predictor of miscarriage risk in our study despite previous concerns about its pulsatile secretion. Low serum progesterone and PIBF levels predicted spontaneous miscarriage among women presenting with threatened miscarriage between gestation weeks 6 to 10. Predictive models to calculate probability of spontaneous miscarriage based on serum progesterone, together with maternal BMI and fetal heart are proposed.

Maternal serum progesterone-induced blocking factor (PIBF) in the prediction of preterm birth.

The objective of this study was to analyze the maternal serum concentration of progesterone-induced blocking factor (PIBF) with regard to the prediction and the interval between sampling and the onset of preterm birth. A prospective study was conducted on a sample of 37 women with threatened pre-term birth and 41 healthy pregnant women between the 24th and 28th gestational weeks. Out of 37 patients with threatened preterm birth 11 delivered pre-term and three groups of patients were formed: the preterm delivery group, patients with threatened preterm delivery, and healthy pregnant women. In samples that were taken within 5 days before labor started (6/11, 54.5%), PIBF concentrations were significantly lower than in those obtained more than 5 days before labor (5/11, 45.5%; the mean interval between sampling and the onset of labor was 4.1 ± 1.8 days). Multiple regression analysis of the individual contributions of each observed parameter for preterm delivery demonstrated the significant contribution of a lack of PIBF to preterm birth (p = 0.002). Receiver operating characteristics (ROC) analysis was performed to evaluate the diagnostic accuracy of PIBF for the prediction of preterm birth of women with symptoms of pre-term delivery. The PIBF demonstrated an excellent diagnostic value in the prediction of preterm birth with an area under the ROC curve (AUC) of 0.956 (95% CI = 0.884-0.989; p < 0.0001). Our data suggest that pregnancy termination can be predicted by lower than normal pregnancy PIBF values within 5 days before labor and can contribute to the diagnosis of preterm birth.

Early pregnancy sex steroids and maternal breast cancer: a nested case-control study.

Pregnancy, parity, and circulating steroid hormone levels are associated with risk of breast cancer, but little is known about hormone concentrations during pregnancy and subsequent breast cancer risk. We evaluated early pregnancy (<140 days gestation) serum estradiol, estrone, progesterone, and testosterone and breast cancer risk in a nested case-control study in the Finnish Maternity Cohort. The cohort includes 98% of pregnancies registered in Finland since 1983. Individuals with samples collected in the first pregnancy leading to a live birth were eligible. Breast cancer cases (n = 1,199) were identified through linkage with the Finnish Cancer Registry; 2,281 matched controls were selected using incidence density sampling. ORs were calculated using conditional logistic regression. Hormone concentrations were not associated with breast cancer overall. Estradiol was positively associated with risk of breast cancer diagnosed age <40 [4th vs. 1st quartile OR 1.60 (1.07-2.39); Ptrend = 0.01], and inversely associated with breast cancer diagnosed at age ≥40 [4th vs. 1st quartile OR 0.71 (0.51-1.00); Ptrend = 0.02]. Elevated concentrations of the steroid hormones were associated with increased risk of estrogen receptor (ER)- and progesterone receptor (PR)-negative tumors in women age <40 at diagnosis. We observed no association between steroid hormones and ER(+)/PR(+) disease. These data suggest a positive association between high concentrations of early pregnancy steroid hormones and risk of ER(-)/PR(-) breast cancer in women diagnosed age <40, and an inverse association for overall breast cancer diagnosed age ≥40. Further research on pregnancy hormones and risk of steroid receptor-negative cancers is needed to further characterize this association.

Mifepristone causing complete remission of rapidly advancing leukemia with measurement of progesterone-induced blocking factor.

BACKGROUND: Mifepristone has been demonstrated to cause palliation from murine and human cancer, even in cancers not known to be positive for expression of progesterone receptors. The aim of the present study was to determine if rapidly advancing chronic lymphocytic leukemia responds to mifepristone therapy, and if so, is this effect related to increased expression of the progesterone-induced blocking factor? CASE REPORT: An 81-year-old woman with chronic lymphocytic leukemia whose condition progressed to the acute rapidly progressing stage agreed to be exclusively treated orally with 200 mg mifepristone daily. RESULTS: The patient showed a dramatic improvement after a short exposure time to mifepristone. Complete remission has persisted so far for 12 months on exclusive mifepristone therapy. Her PIBF levels were normal before mifepristone therapy and did not change after treatment. CONCLUSION: Mifepristone can provide marked improvement of human leukemia even in the absence of increased serum PIBF levels.

Increased circulating myeloid-derived suppressor cells correlated negatively with Th17 cells in patients with rheumatoid arthritis.

OBJECTIVES: Myeloid-derived suppressor cells (MDSCs) have recently been identified as an important mediator in inflammatory and autoimmune diseases through the production of arginase (Arg)-1 and inducible nitric oxide synthase (iNOS). The aim of this study was to investigate the prevalence of MDSCs in the peripheral blood of patients with rheumatoid arthritis (RA) and evaluate their correlation with T-helper (Th)17 cells. METHOD: The frequency of MDSCs and Th17 cells and the mRNA expression of transcriptional factor RORγ-t and iNOS in the peripheral blood of RA patients and healthy controls (HC) were determined by flow cytometry and real-time reverse transcription polymerase chain reaction (RT-PCR), respectively. Plasma levels of interleukin (IL)-17, IL-6, tumour necrosis factor (TNF)-α, and Arg-1 were analysed by enzyme-linked immunosorbent assays (ELISA). RESULTS: Compared with HC, both the prevalence of circulating MDSCs and plasma Arg-1 increased significantly in RA patients. However, no significant difference was observed in the mRNA level of iNOS between RA patients and HC. The frequency of Th17 cells in RA patients was significantly higher than in HC but correlated negatively with the frequency of MDSCs and plasma Arg-1. A negative correlation between MDSCs and plasma TNF-α was also observed. However, the frequency of MDSCs was not correlated with plasma IL-6 and IL-17, nor with the mRNA level of RORγ-t. CONCLUSIONS: We found a negative correlation between increased circulating MDSCs and Th17 cells in RA patients, which may provide new insights into the mechanisms involved in RA.

Dydrogesterone supplementation in women with threatened preterm delivery--the impact on cytokine profile, hormone profile, and progesterone-induced blocking factor.

Progesterone is indispensable in creating a suitable endometrial environment for implantation, and also for the maintenance of pregnancy. Successful pregnancy depends on an appropriate maternal immune response to the fetus. A protein called progesterone-induced blocking factor (PIBF) acts by inducing Th2-dominant cytokine production to mediate the immunological effects of progesterone. The aim of this prospective study was to compare serum concentrations of progesterone (P), estradiol (E2), anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNFα, IFNγ) cytokines, and serum PIBF concentrations in women with threatened preterm delivery who were given progesterone supplementation (study group) with those of women with threatened preterm delivery who were not given progesterone supplementation (control group). After dydrogesterone treatment of patients in the study group, serum PIBF as well as progesterone concentrations significantly increased. Women in this group had significantly higher serum levels of IL-10 than controls. The length of gestation was significantly higher in the group of women who were given progesterone supplementation. Our data suggest that dydrogesterone treatment of women at risk of preterm delivery results in increased PIBF production and IL-10 concentrations, and lower concentrations of IFNγ.

Maternal serum progesterone-induced blocking factor at 11-13 weeks' gestation in spontaneous early preterm delivery.

OBJECTIVE: Progesterone-induced blocking factor (PIBF) may be the mediator of the pregnancy maintenance effects of progesterone. The aim of this study is to investigate the potential value of measuring the maternal serum concentration of PIBF at 11-13 weeks' gestation in the prediction of spontaneous early preterm delivery. METHOD: The maternal serum concentration of PIBF at 11-13 weeks was measured by enzyme-linked immunosorbent assay in 25 singleton pregnancies which subsequently delivered spontaneously before 34 weeks, and 75 controls who delivered at or after 37 weeks. The values in the 2 groups were compared by the Mann-Whitney U test. RESULTS: The median maternal serum concentration of PIBF in women who subsequently delivered before 34 weeks (157.5, interquartile range 99.5-208.8 ng/ml) was not significantly different from the control group delivering at term (167.5, interquartile range 105.0-212.0 ng/ml; p = 0.519). CONCLUSIONS: In women who have a spontaneous early preterm delivery, the maternal serum levels of PIBF are not altered at 11-13 weeks of gestation.

A practical approach to the prevention of miscarriage: part 2--active immunotherapy.

PURPOSE: To present data suggesting that active immunization with lymphocyte immunotherapy is a treatment that has benefit in preventing miscarriage. METHODS: Lymphocyte immunotherapy is given to women with a history of recurrent miscarriage or failure to achieve a successful pregnancy, despite several previous embryo transfers. Active immunization was combined with progesterone therapy. The lymphocytes were not refrigerated, but used fresh. RESULTS: Compared to controls, i.e., progesterone therapy alone, the injection of paternal lymphocytes intradermally improved miscarriage rates and improved live delivered pregnancy rates per embryo transfer. CONCLUSIONS: The addition of progesterone treatment may act synergistically with lymphocyte immunotherapy, especially in primary aborters and tertiary aborters. However, it is important to use fresh--not refrigerated--stored lymphoctes.