An economic analysis of preimplantation genetic testing for aneuploidy by polar body biopsy in advanced maternal age.

OBJECTIVE: What are the cost per live birth and the incremental cost of preventing a miscarriage with preimplantation genetic testing for aneuploidy (PGT-A) by polar body biopsy and array-based comprehensive genome hybridisation (aCGH) versus regular IVF/ICSI without PGT-A for infertility treatment in women 36-40 years of age? DESIGN: Decision tree model. POPULATION: A randomised clinical trial on PGT-A (ESTEEM study). METHODS: Two treatment strategies were compared: one cycle of IVF/ICSI with or without PGT-A. Costs and effects were analysed with this model for four different cost scenarios: high-, higher medium, lower medium and low-cost. Base case, sensitivity, threshold, and probabilistic sensitivity analyses were used to examine the cost-effectiveness implications of PGT-A. RESULTS: PGT-A increased the cost per live birth by approximately 15% in the high-cost scenario to approximately 285% in the low-cost scenario. Threshold analysis revealed that PGT-A would need to be associated with an absolute increase in pregnancy rate by 6% to >39% or, alternatively, would need to be US$2,969 (high-cost scenario) to US$4,888 (low-cost scenario) cheaper. The incremental cost to prevent one miscarriage by PGT-A using the base case assumptions was calculated to be US$34,427 (high-cost scenario) to US$51,146 (low-cost scenario). A probabilistic sensitivity analysis showed cost-effectiveness for PGT-A from 1.9% (high-cost scenario) to 0.0% (low-cost scenario) of calculated samples. CONCLUSIONS: While avoiding unnecessary embryo transfers and miscarriages are important goals, patients and doctors need to be aware of the high-cost implications of applying PGT-A using aCGH on polar bodies. TWEETABLE ABSTRACT: PGT-A by polar body biopsy and comprehensive genome hybridisation increases cost per live birth and requires high financial spending per miscarriage averted.

Serum anti-Müllerian hormone concentration and follicle density throughout reproductive life and in different diseases-implications in fertility preservation.

STUDY QUESTION: How do anti-Müllerian hormone (AMH) serum concentrations and follicle densities (FDs) change with age and disease and what are the implications for fertility preservation? SUMMARY ANSWER: AMH concentrations and FD do not correlate in young women, and AMH but not FD is reduced in some diseases, limiting the value of AMH as a predictive parameter of ovarian tissue transplantation. WHAT IS KNOWN ALREADY: AMH is widely used as a parameter to estimate the ovarian reserve. However, the reliability of AMH to predict total number of follicles and the FD is questionable. Women with lymphoma and leukaemia have been shown to have reduced AMH concentrations, but it is unknown if the FD is also reduced. In fertility preservation it is essential to estimate the correct total number of follicles and the FD, as ovarian tissue should only be cryopreserved if ovarian reserve is high. Furthermore, the amount of tissue to be transplanted should be based on the estimation of the real FD. STUDY DESIGN, SIZE, DURATION: This retrospective observational study included 830 women (mean ± SD age, 28.2 ± 6.81 years; range, 4-43 years) with malignant (n = 806) and benign (n = 24) diseases who cryopreserved tissue in a single centre as part of a national fertility preservation programme. Females with ovarian surgery or known predispositions for a reduced ovarian reserve were excluded. AMH concentrations and FD were evaluated from March 2011 to September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMH concentrations were analysed before gonadotoxic therapies. Standardized biopsies, obtained from different areas of ovarian cortex, were collected. FD was analysed after tissue digestion and calcein staining and was expressed as average number of primordial and primary follicles count per 3 mm biopsy and per cubic millimeter tissue. AMH concentrations and FD were analysed in relation to age and diagnosis group. Both parameters were age adjusted, and associations between the different diagnosis groups and AMH versus FD were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Mean ± SD AMH concentration was 3.1 ± 2.81 g/ml, mean FD per 3 mm biopsy was 137 ± 173.9 and 19.4 ± 24.60 per mm3. Maximum AMH concentrations were found in children and teenagers at the age of 6-10 years (5.71 ng/ml) and in adults at the age of 21-25 years (3.33 ng/ml). FD was highest in young children up to an age of 15 years and decreased with increasing age. AMH and FD were not correlated in women ≤20 years and weakly to moderately correlated in women 21-40 years (r = 0.24-0.39). Age-adjusted correlations between AMH and FD were demonstrated in several diagnosis groups such as breast cancer, leukaemia, sarcoma, gastrointestinal cancer and gynaecological cancer but not in the groups exhibiting Hodgkin's and non-Hodgkin's lymphoma, cerebral cancer, other types of malignancies and other types of benign diseases. Further statistical analysis supported the finding that, in some diagnosis groups such as Hodgkin's lymphoma and in gynaecological cancer, AMH concentrations but not FDs are reduced, questioning the prognostic accuracy of AMH for the FD in these diseases. LIMITATIONS, REASONS FOR CAUTION: Even though biopsies were taken from different sites, heterogenous distribution of follicles might have had some effect on the accuracy of the analysis. WIDER IMPLICATION OF THE FINDINGS: AMH should be used with care to estimate the total ovarian reserve and FD of cancer patients in young women in some diseases. Therefore, calculating the amount of ovarian tissue to be transplanted based solely on AMH might be inaccurate whereas FD might be a better parameter. STUDY FUNDING/COMPETING INTEREST(S): The study did not receive any exterior funding.

MIR137 variants identified in psychiatric patients affect synaptogenesis and neuronal transmission gene sets.

Sequence analysis of 13 microRNA (miRNA) genes expressed in the human brain and located in genomic regions associated with schizophrenia and/or bipolar disorder, in a northern Swedish patient/control population, resulted in the discovery of two functional variants in the MIR137 gene. On the basis of their location and the allele frequency differences between patients and controls, we explored the hypothesis that the discovered variants impact the expression of the mature miRNA and consequently influence global mRNA expression affecting normal brain functioning. Using neuronal-like SH-SY5Y cells, we demonstrated significantly reduced mature miR-137 levels in the cells expressing the variant miRNA gene. Subsequent transcriptome analysis showed that the reduction in miR-137 expression led to the deregulation of gene sets involved in synaptogenesis and neuronal transmission, all implicated in psychiatric disorders. Our functional findings add to the growing data, which implicate that miR-137 has an important role in the etiology of psychiatric disorders and emphasizes its involvement in nervous system development and proper synaptic function.

Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF.

Bemfola (follitropin alfa) (Finox AG, Switzerland), a new recombinant FSH, has a comparable pharmacological profile to that of Gonal-f (Merck Serono, Germany), the current standard for ovarian stimulation. A randomized, multi-centre, Phase 3 study in women undergoing IVF or intracytoplasmic sperm injection (n = 372) showed Bemfola yielding similar efficacy and safety profiles to Gonal-f. Women aged 20-38 years of age were randomized 2:1 to receive a single, daily, subcutaneous 150 IU dose of either Bemfola or Gonal-f. This study tested equivalence in the number of retrieved oocytes using a pre-determined clinical equivalence margin of ±2.9 oocytes. Compared with Gonal-f, Bemfola treatment resulted in a statistically equivalent number of retrieved oocytes (Bemfola 10.8 ± 5.11 versus Gonal-f 10.6 ± 6.06, mean difference: 0.27 oocytes, 95% confidence interval: -1.34, 1.32) as well as a similar clinical pregnancy rate per embryo transfer in first and second cycles (Bemfola: 40.2% and 38.5%, respectively; Gonal-f: 48.2% and 27.8%, respectively). No difference in severe ovarian hyperstimulation syndrome was observed between treatment groups (Bemfola: 0.8%; Gonal-f: 0.8%). This study demonstrates similar clinical efficacy and safety profiles between Bemfola and Gonal-f, and suggests that Bemfola can be an appropriate alternative in ovarian stimulation protocols.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Fertility and gonadal function in female survivors after treatment of early unfavorable Hodgkin lymphoma (HL) within the German Hodgkin Study Group HD14 trial.

BACKGROUND: In the HD14 trial, 2×BEACOPPescalated+2×ABVD (2+2) has improved the primary outcome. Compared with 4×ABVD, this benefit might be compromised by more infertility in women. Therefore, we analyzed gonadal function and fertility. PATIENTS AND METHODS: Women≤45 years in ongoing remission at least 1 year after therapy were included. Hormone parameters, menopausal symptoms, measures to preserve fertility, menstrual cycle, pregnancies, and offspring were evaluated. RESULTS: Three hundred and thirty one of 579 women addressed participated (57.2%) and 263 per-protocol treated patients qualified (A=ABVD: 137, B=2+2: 126, mean time after therapy 42 and 43 months, respectively). Regular menstrual cycle after treatment (A: 87%, B: 83%) and time to recovery (≤12 months) were not different. Follicle-stimulating hormone and anti-Muellerian hormone were significantly better in arm A. However, pregnancies after therapy favored arm B (A: 15%, B: 26%, P=0.043) and motherhood rates were equivalent to the German normal population. Multivariate analysis revealed prophylactic use of gonadotropin-releasing hormone (GnRH) analogues as highly significant prognostic factor for preservation of fertility (odds ratio=12.87, P=0.001). Severe menopausal symptoms were frequent in women≥30 years (A: 21%, B: 25%). CONCLUSIONS: Hormonal levels after 2+2 indicate a reduced ovarian reserve. However, 2+2 in combination with GnRH analogues does not compromise fertility within the evaluated observation time.

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation.

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim-up technique and incubated either at room temperature or at 37 °C for up to 4 h. The presence of sperm nuclear vacuoles was determined by contrast-enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6 ± 17.6%; 37 °C: 48.4 ± 17.0%) following 4 h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim-up or washed and incubated at 37 °C. After 4 h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5 ± 15.4% to 68.6 ± 9.0%; P < 0.05) but not in swim-up sperm (from 51.5 ± 15.4% to 48.2 ± 17.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37 °C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim-up.

Environmental pollutants and type 2 diabetes: a review of mechanisms that can disrupt beta cell function.

The prevalence of diabetes mellitus is currently at epidemic proportions and it is estimated that it will increase even further over the next decades. Although genetic predisposition and lifestyle choices are commonly accepted reasons for the occurrence of type 2 diabetes, it has recently been suggested that environmental pollutants are additional risk factors for diabetes development and this review aims to give an overview of the current evidence for this. More specifically, because of the crucial role of pancreatic beta cells in the development and progression of type 2 diabetes, the present work summarises the known effects of several compounds on beta cell function with reference to mechanistic studies that have elucidated how these compounds interfere with the insulin secreting capacity of beta cells. Oestrogenic compounds, organophosphorus compounds, persistent organic pollutants and heavy metals are discussed, and a critical reflection on the relevance of the concentrations used in mechanistic studies relative to the levels found in the human population is given. It is clear that some environmental pollutants affect pancreatic beta cell function, as both epidemiological and experimental research is accumulating. This supports the need to develop a solid and structured platform to fully explore the diabetes-inducing potential of pollutants.

Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening.

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.

Toxicity evaluation of perfluorooctane sulfonate (PFOS) in the liver of common carp (Cyprinus carpio).

Perfluorooctane sulfonate (PFOS) has been manufactured for over 50 years in increasing quantities and has been used for several industrial and commercial aims. Due to persistence and bioaccumulation of this pollutant, it can be found worldwide in wildlife and humans. Biochemical effects of PFOS exposure are mainly studied in mammalian model species and information about effects on fish species remain largely scarce. This lack of toxicity data points out that there is an urgent need for the mechanistic molecular understanding of the mode of action of this pollutant. In the present study, common carp (Cyprinus carpio) was exposed through water for 14 days at concentrations of 0.1, 0.5 and 1 mg/l PFOS. Liver was selected as target tissue. Custom microarrays were constructed from cDNA libraries obtained with Suppression Subtractive Hybridization-Polymerase Chain Reaction (SSH-PCR) experiments. Microarray data revealed that the expression of several genes in the liver was influenced by PFOS exposure and real-time PCR was used to confirm these gene expression changes. The affected genes were mainly involved in energy metabolism, reproduction and stress response. Furthermore, the relative condition factor, the hepatosomatic index, and the available glycogen reserves of the exposed fish were significantly lower after 14 days of exposure than in the control fish. At all levels of biological organization, indications of a trade-off between the metabolic cost of toxicant exposure on one hand and processes vital to the survival of the organism on the other hand were seen. Our results support the prediction that increases in energy expenditure negatively affects processes vital to the survival of an organism, such as growth.

Swimming performance and energy metabolism of rainbow trout, common carp and gibel carp respond differently to sublethal copper exposure.

We compared the effects of sublethal waterborne copper exposure on swimming performance and respiration rates in rainbow trout, Oncorhynchus mykiss, with those in less sensitive cyprinid species such as common carp, Cyprinus carpio, and gibel carp, Carassius auratus gibelio. These cyprinids are considerably more resistant to Cu intoxication, and differ from trout in swimming performance and respiratory behaviour. Critical swimming speed (U(crit)), oxygen consumption, plasma ammonia and muscle ammonia, lactate and pH were measured during a 28-day sublethal exposure to 1 microM Cu. U(crit) decreased with 48, 31 and 13% within the first 12-24 h for rainbow trout, common and gibel respectively. Gibel carp recovered quickly and experienced no further reduction in swimming performance. Recovery of swimming capacity in rainbow trout and common carp was only partial. All three species displayed similar plasma ammonia peaks in the first hours to days, and a more gradual muscle ammonia accumulation over time. Whereas no signs of respiratory stress occurred in rainbow trout, common carp experienced a transient reduction in oxygen consumption combined with anaerobic metabolism after 24 h of exposure. At the same time, oxygen consumption was also reduced in gibel carp, but no signs of anaerobic metabolism were detected. Cu accumulated quickly to similar levels (36-39 microg g(-1) dry weight at day 3) in the gills of all three species, after which accumulation leveled off. Liver tissue of rainbow trout had a high Cu level from the start, and Cu concentration did not show any additional accumulation. In contrast, common carp liver showed a significant Cu accumulation from day 3 onwards, while accumulation in gibel livers was much slower and was significant from day 7 onwards. Interestingly, Cu accumulation patterns in plasma and kidney revealed a possibly important role for the kidney in Cu homeostasis of gibel carp.

Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA).

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.

HLA-G polymorphisms and allele frequencies in Caucasians.

HLA-G, a nonclassical class I MHC molecule, is uniquely expressed on extravillous cytotrophoblasts of the maternal-fetal interface and is suggested to be essential for establishment of maternal-fetal immune tolerance. Although the level of polymorphism in HLA-G has originally been considered low, number, nature and site of polymorphisms seem to vary between different ethnic populations. We investigated HLA-G polymorphisms in a population of German and Croatian origin by SSCP-analysis and direct sequencing as well as RFLP analysis for presence of the 1597delC mutation. HLA-A alleles associated with the different HLA-G alleles were determined by SSP PCR-typing. In Caucasians, HLA-G exhibits a low degree of polymorphism on the amino-acid level and only slightly higher variability on the nucleotide level. In 264 independent chromosomes, 4 HLA-G alleles on the level of amino acid polymorphisms and an additional 6 variations of nucleotide sequences could be identified. The null-allele G*0105N was present at an allele frequency of 2.3%, which is higher than initially suggested for Caucasians but lower than in Hispanics and African-Americans. Furthermore, some HLA-G alleles exhibit strong linkage disequilibrium with HLA-A.

Soluble HLA levels in early pregnancy after in vitro fertilization.

Intact pregnancy can be interpreted as a state of maternal immunotolerance toward an haploidentical fetus. Soluble HLA (sHLA) molecules increase during episodes of allograft rejection and are discussed as candidates to modulate immune responses. We questioned whether after in vitro fertilization (IVF) the subsequent intact pregnancy, early abortion, or tubal pregnancy influence the courses sHLA serum levels. Therefore, serum samples of 65 IVF patients were assayed by ELISA for sHLA-I, sHLA-G, and sHLA-DR concentrations preovulatorily and after a positive HCG test weekly until the 9th gestational week (GW). In 20 patients experiencing an early abortion the preovulatory sHLA-G mean level of 25.9 +/- 3.9 SEM ng/ml and the share of 4.2 +/- 0.8 SEM % on total sHLA-I were significantly (p < 0.05) reduced compared to women with intact pregnancy. The same differences (p < 0.0001) were seen during the monitoring of sHLA-G and sHLA-I levels in intact pregnancy versus early abortion until 9th GW. Twin pregnancy revealed a drastically increase of sHLA-G levels from the 8th GW compared to singleton pregnancies. Further, individual sHLA-DR levels increased during intact pregnancy but decreased in the group of early abortion. With regard to sensitivity and specificity for pregnancy outcome sHLA quantitation reached similar weight as routine HCG determinations at GW 5. Especially women with preovulatory low sHLA-G levels appear to be on risk for early abortion after IVF.

HLA-G polymorphisms and molecule function--questions and more questions--a review.

Human leukocyte antigen (HLA)-G is a non-classical HLA-class I antigen which is predominantly expressed on invasive trophoblastic cells and is postulated to be a mediator of maternal-fetal tolerance. HLA-G interacts with NK cells, can present nonamer peptides and binds CD8 in an analogous manner to classical HLA-I. The HLA-G protein exists in soluble and membrane-bound isoforms generated through alternative splicing. Although initially considered to be non-polymorphic, variations of the HLA-G DNA sequence have been reported which led to the definition of a limited number of HLA-G alleles including the Null-allele G*0105N. Whereas the HLA-G DNA sequence shows a high degree of conservation in positions which are essential for classical HLA-I molecule functions, polymorphic sites in HLA-G are not congruent with sites of high nucleotide variability in classical HLA. The identification of two females with recurrent spontaneous abortions who are homozygous for the G*0105N Null-allele re-opens the discussion about the role of HLA-G in pregnancy and underlines the need of a systematic analysis of the different hypotheses of HLA-G function in vivo.

HLA-DQA1 and HLA-DQB1 haplotypes in aborted fetuses and couples with recurrent spontaneous abortion.

HLA haplotypes may be associated with spontaneous abortion through a variety of mechanisms, including maternal hyporesponsiveness to fetal alloantigens, maternal autoimmunity, and HLA-linked t-locus homologues. HLA-DQA1 and HLA-DQB1 haplotypes were determined in 37 couples with a history of recurrent spontaneous abortion (RSAB), 40 of their abortuses, and 20 fertile control couples. The distribution of haplotype frequencies did not differ between control subjects and RSAB wives, RSAB husbands, or abortuses. The frequency of the HLA-DR5-linked haplotype, DQA1*501/DQB1*301, which was considered a marker for immune hyporesponsiveness, did not differ between RSAB wives and control subjects (P = 0.353). The frequency of the autoimmune-associated HLA-DR3-linked haplotype, DQA1*0501/DQB1*0201, did not differ significantly between RSAB wives and control subjects (P = 0.103). The frequency of the DQA1*0201/DQB1*0201 haplotype in RSAB husbands was greater than the 95th percentile confidence limit of the frequency of this haplotype in control subjects. Among seven RSAB husbands who were heterozygous for this haplotype and did not share a DQA1*0201 allele with his wife, the haplotype was transmitted to 6 of 7 abortuses (3.5 expected). Although the small size of this sample precludes drawing conclusions regarding HLA transmission biases in RSAB couples, these data have generated a specific hypothesis regarding the DQA1*0201/DQB1*0201 haplotype that can be investigated in future studies.

Population genetic studies of HLA-G: allele frequencies and linkage disequilibrium with HLA-A1.

HLA-G is a class I gene that is expressed in the extravillous cytotrophoblast. Although the function of this gene is still unknown, its expression at the maternal-fetal interface suggests that HLA-G may play a key role in the induction of tolerance during pregnancy. Preliminary to our studies of the effects of HLA-G polymorphisms on pregnancy outcome, we have defined HLA-G alleles in the Hutterites. We report here the presence of nine HLA-G alleles that differ with respect to nucleotide sequences, including four groups of alleles that differ with respect to amino acid sequences, and striking linkage disequilibrium between HLA-G and HLA-A alleles. The levels and sites of polymorphism in HLA-G suggest that this gene had a unique evolutionary history and may perform nonclassical functions at the maternal-fetal interface.