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// Lindsay F. Goodale 1,6 , Soren Hayrabedran 2 , Krassimira Todorova 2 , Roumen Roussev 3 , Sivakumar Ramu 3,8 , Christopher Stamatkin 3,9 , Carolyn B. Coulam 3 , Eytan R. Barnea 4,5,* and Robert O. Gilbert 1,7,* 1 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA 2 Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria 3 CARI Reproductive Institute, Chicago, IL, USA 4 BioIncept, LLC, Cherry Hill, NJ, USA 5 Society for the Investigation of Early Pregnancy (SIEP), Cherry Hill, NJ, USA 6 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA, USA 7 Ross University School of Veterinary Medicine, Basseterre, St. Kitts, West Indies 8 Promigen Life Sciences, Downers Grove, IL, USA 9 Therapeutic Validation Core, Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Eytan R. Barnea, email: // Keywords : recurrent pregnancy loss, preImplantation factor (PIF), oxidative stress, PDI, embryo, Autophagy Received : December 05, 2016 Accepted : February 22, 2017 Published : March 08, 2017 Abstract Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos’ development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by 2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor’s pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

Recurrent pregnancy loss, defined as two or more failed pregnancies by the American Society for Reproductive Medicine [1], is a clinical problem because known effective treatment is limited. Effective treatment depends on the cause of the reproductive failure. Thus, attention has been focused on determining causes of recurrent pregnancy loss. Even so, to date, only chromosomal abnormalities of the conceptus and immunologic risk factors have been generally accepted as etiologies of recurrent pregnancy loss. Recently, a novel pathologic pathway that involves impaired decidualization of endometrial stromal cells has been proposed as a cause of recurrent pregnancy loss [2]. Further, it has been hypothesized that the impaired decidualization is manifested by the prolongation of the window of implantation allowing for increased fecundity and Bsuperfertility^ [2–7]. The following paragraphs will describe the evidence for these notions.

Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo development in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25ng/ml (37% and 42%) compared with PIFscr (18%; P

Problem To search for molecular markers of endometriosis the following polymorphisms: p53 codon 72 Pro (apoptosis), TNF alpha-308 (inflammation), VEGF-1164AA (angiogenesis), and SOD2 (oxidative stress) were investigated. Method of study Forty-two women—24 with surgically proven endometriosis and 18 with no endometriosis found at the time of laparoscopy–had buccal swabs taken for DNA analyses of 4 gene polymorphisms including p53codon72, TNF-308 G/A, VEGF-1154G/A, SOD Ala16Val DNA. The frequencies of genotypes and alleles of these polymorphisms were compared between women with and without endometriosis. Results No specific gene mutation differences for the four genes tested nor differences in the frequencies of heterozygous and homozygous mutations were found between patients with endometriosis and controls. In addition, no differences in allelic frequencies of the four genetic polymorphisms were observed between patients with endometriosis and control. Conclusion Endometriosis is not associated with gene mutations for p53codon72, TNF-308 G/A, VEGF-1154G/A, SOD Ala16Val.

Problem To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. Method of study Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. Results Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. Conclusion HHV-6 infection is an important factor in RIF.

Problem To determine whether the ACE D/D genotype or the combination of PAI-1 4G/4G and ACE D/D genotypes may serve as a risk factor for recurrent pregnancy loss. Method of study Buccal swabs were obtained from 120 women experiencing recurrent pregnancy loss and from 84 fertile control women. DNA was extracted from the buccal swab samples using the Qiagen DNA Mini Kit (Qiagen), followed by multiplex polymerase chain reaction (PCR). PCR products were analyzed for the ACE gene polymorphism, which consists of the insertion or deletion (I/D) of a 287-bp fragment in intron 16, and the PAI-1 4G/4G genotype. Results No significant differences in specific ACE gene mutations were observed when patients experiencing recurrent miscarriage were compared with control women. When the frequencies of homozygous mutations for ACE D/D and PAI-I 4G/4G were compared between recurrent aborters and controls, again no significant differences in the prevalence of the combination of these gene mutations were noted. Conclusion Homozygosity for the D allele of the ACE gene and the combination of the D/D genotype with two 4G alleles of the PAI-1 promoter gene are not associated with a significant increase in the risk of recurrent miscarriage.

Objective To determine if soluble human leukocyte antigen-G (sHLA-G) concentrations in spent culture media may assist in identifying the normal embryo for implantation. Design Prospective blinded comparative study. Setting Reproductive genetic and reproductive medicine centers. Patient(s) One hundred and sixteen embryos obtained from eight patients undergoing in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD). Intervention(s) Culture media obtained 2 days after fertilization were analyzed for sHLA-G concentrations using an enzyme-linked immunosorbent assay (ELISA) assay. A sHLA-G concentration of ≥1.9 mIU/mL was considered a positive predictor for successful implantation. Polar bodies and blastomeres from day-3 embryos were tested by PGD for 5 to 11 chromosomes: 8, 9, 13, 15, 16, 17, 18, 21, 22, X, and Y. Main Outcome Measure(s) The results of the sHLA-G concentrations were compared with the results of the PGD analyses. Result(s) We found an sHLA-G concentration ≥1.9 mIU/mL in 48% (56 out of 116) and normal PGD results in 52% (57 out of 116) of embryos. Of the embryos with normal PGD results, 46% (26 out of 57) had sHLA-G concentrations ≥1.9 mIU/mL. Among the embryos with sHLA-G ≥1.9 mIU/mL, 46% (26 out of 56) had normal PGD results, and 21% of embryos displayed both normal PGD results and sHLA-G ≥1.9 mIU/mL. Conclusion(s) No correlation between concentrations of sHLA-G in embryo culture media and PGD results of an embryo's aneuploidy were observed.

Problem We have previously reported the role of polymorphisms of thrombogenic genes involved in coagulation and fibrinolysis as risk factors for recurrent pregnancy loss. Thrombophilia has been viewed as a multigenic disorder rather than a monogenetic clinical phenotype and Apo E has been shown to play an important role in lipid metabolism in pregnancy. As individuals carrying the E4 allele of the ApoE gene have the highest risk for thrombosis, we evaluated the frequency of the Apo E4 genotype among women suffering from recurrent pregnancy loss. Method of study Buccal swabs were obtained from 69 women with a history of two or more consecutive spontaneous abortions and 37 women with at least two live births and not more than one miscarriage. DNA was extracted from the buccal swabs and PCR amplification of Apo E2, E3, and E4 was performed. Results Women experiencing recurrent pregnancy loss had a significantly higher prevalence of Apo E3/4, E4/4 genotypes (21.7%) compared with control women (5.4%) (P = 0.036). Conclusion Apo E4 polymorphism may contribute to the thrombophilic risk factors contributing to recurrent pregnancy loss.

Problem Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls. Method of study Six hundred thirty-four participants including 550 women with a history of recurrent pregnancy loss, 43 individuals with deep vein thrombosis and 41 fertile women without a history of recurrent miscarriage. All participants had buccal swabs taken for DNA analyses of nine gene polymorphisms including factor V G1691A, factor V H1299R (R2), factor II Prothrombin G20210A, factor XIII V34L, β-fibrinogen −455G>A, PAI-1 4G/5G, human platelet antigen 1 a/b (L33P), MTHFR C677T, MTHFR A1298C. Frequencies of thrombophilic gene polymorphisms were compared among the three populations studied. Results Individuals with a history of DVT had a significantly higher frequency of all of the polymorphisms studied compared with women experiencing a history of recurrent pregnancy loss and the fertile controls. The frequencies of mutations for V34L and PAI-1 4G/5G were significantly increased among women experiencing recurrent pregnancy loss compared with controls. The most prevalent polymorphisms were factor XIII V34L and PAI-1 4G/4G for both individuals with a history of deep vein thrombosis and recurrent pregnancy loss compared with controls. Conclusion Screening for risk factors for inherited thrombophilia with only polymorphisms for factor V von Leiden, factor II prothrombin and MTHFR may be missing the more prevalent identifiers of jeopardy.

Rinehart Center for Reproductive Medicine, Chicago, IL, USAKeywordsIntralipid, NK cell activity, suppressionCorrespondenceRoumen Roussev, 233 East Erie St, Suite 510,Chicago, IL 60611, USA.E-mail: rgroussev@yahoo.comSubmitted March 25, 2008;accepted April 25, 2008.CitationRoussev RG, Acacio B, Ng SC, Coulam CB.Duration of intralipid’s suppressive effect onNK cell’s functional activity. Am J ReprodImmunol 2008; 60: 258–263doi:10.1111/j.1600-0897.2008.00621.x

Problem The interleukin-1 system has been implicated in pregnancy outcome. Fetal carriage of interleukin-1 receptor antagonist (IL-1Ra) specific alleles has been associated with adverse pregnancy outcomes including spontaneous abortion and pre-term labor. This study was undertaken to compare the frequency of IL-1RN*2 alleles among both male and female partners of couples experiencing recurrent pregnancy loss with that of fertile control couples. Method of study Buccal swabs were obtained from 42 couples experiencing recurrent pregnancy loss and from 20 fertile control couples. DNA was extracted from the buccal swabs and analyzed for the presence of IL-1RN variable number tandem repeat. Results No significant differences were found when the frequency of IL-1RN*2 polymorphisms were compared between fertile control couples and couples experiencing recurrent pregnancy loss. Similar results were also obtained when comparing women or men respectively from each group. Conclusion IL-1RN*2 allele is not a risk factor for recurrent pregnancy loss.

Problem To be successful, pregnancy must induce its own blood supply through angiogenesis and vascular endothelial growth factor (VEGF) is the best characterized regulator of angiogenesis and one polymorphism of the VEGF gene, -1154, has been suggested to be associated with recurrent spontaneous abortion. The aim of this study was to confirm or refute the relationship of VEGF -1154 to recurrent pregnancy loss (RPL). Method of study Buccal swabs were obtained from 152 women with history of two or more consecutive spontaneous abortions and 65 control women. DNA was extracted from the buccal swabs and analyzed for the presence of the VEGF -1154A/A gene. Results The frequency of homozygosity of the VEGF -1154A gene was significantly higher among women experiencing RPL compared with fertile control women (16% versus 6%, P Conclusion Homozygosity of the VEGF -1154A gene may serve as a susceptibility factor affecting for RPL.

Human leukocyte antigen G (HLA-G) is thought to play a key role in implantation by modulating cytokine secretion to control trophopblastic cell invasion and to maintain a local immunotolerance.The literature is reviewed to provide a description of the genetic background, properties of the protein, and the function of HLA-G. Data are presented on potential clinical applications of HLA-G including the use of evaluation of HLA-G gene polymorphisms in the diagnosis of patients experiencing recurrent pregnancy loss and evaluation and testing of soluble HLA-G (sHLA-G) in embryo culture media for the selection of embryos for transfer after in vitro fertilization (IVF).The literature supports a central role of HLA-G for successful implantation. Of couples experiencing recurrent pregnancy loss, 32% demonstrated the -1725G HLA-G polymorphism. Our data showed that when embryos were selected for transfer after IVF based on culture media concentrations of sHLA-Gor = 2 U/ml and good morphologic grade, a 65% pregnancy rate compared with a 0% pregnancy rate in those with2 U/ml sHLA-G.HLA-G is important for successful implantation in human beings. The HLA-G -725 promoter polymorphism is a risk factor for recurrent miscarriage. Measurement of sHLA-G in embryo culture media can help select embryos for transfer after IVF allowing fewer embryos to be transferred in an attempt to lower multiple gestation rates.

Problem The purpose of this study was to compare the ability of intravenous immunoglobulin (IVIg), intralipid and soluble human leukocyte antigen (sHLA)-G to suppress natural killer (NK) cell cytotoxicity in an in vitro assay. Method of study Blood samples taken from 275 women experiencing reproductive failure were analyzed for NK cytotoxicity and the suppression of NK cytotoxicity by IVIg 4 and 2 mg/mL (n = 275), intralipid 18 and 9 mg/mL (n = 275) and sHLA-G 70 and 35 ng/mL (n = 50) using immunofluorescent labeled K562 cells as targets and flow cytometry. Results Natural killer cytotoxicity was suppressed in all samples. Among patients with normal NK cell activity, IVIg suppressed NK cytotoxicity by 44.9 ± 8.1%, intralipid suppressed NK killing by 45.2 ± 8.3% and sHLA-G suppressed by 49.0 ± 9.2%. When specimens with abnormal NK activity were observed for suppression of cytotoxicity, IVIg suppressed by 38.9 ± 5.4%, intralipid suppressed by 39.8 ± 6.2% and sHLA-G suppressed by 39.9 ± 5.0%. Conclusion Intravenous immunoglobulin, intralipid and sHLA-G suppressed NK cell cytotoxicity with equal efficacy in an in vitro assay.

Objective To determine the prevalence of Y-chromosome microdeletions in recurrent pregnancy loss (RPL) couples as compared with couples with male factor infertility and fertile couples. Design Controlled clinical study. Setting Andrology laboratory and RPL clinic. Patient(s) Seventeen men from RPL couples, 18 men from couples with a live birth and no history of miscarriages, and 10 men from couples with male factor infertility. Intervention(s) Buccal smears for Y-chromosome microdeletion testing. Main Outcome Measure(s) The DNA was tested for microdeletions in the proximal AZFc region by polymerase chain reaction (PCR). Result(s) Fourteen of the 17 men (82%) tested had microdeletions in one or more of the four segments studied. Two of the 10 male factor infertility patients (20%) had microdeletions in 2 different segments. None of the 18 fertile men had any microdeletions in the 4 segments of the proximal AZFc region studied. Conclusion(s) The prevalence of the Y-chromosome microdeletions in the proximal AZFc region was much higher in men from RPL couples than from fertile or infertile couples. Although these patients are from a tertiary referral center that may skew the population and findings, one may consider Y-chromosome microdeletion testing particularly of the AZFc region in the evaluation of RPL couples when all other tests fail to reveal the etiology.

Problem: Implantation determines success of in vitro fertilization (IVF) and embryo transfer (ET) cycles. Data are accumulating to support a role of the immune system in implantation. Most of the literature addresses the importance of natural killer (NK) cells in this process. The purpose of the current study is to examine the role of circulating T cells in implantation failure. Method of study: Blood from 22 women undergoing IVF/ET during November, 2001, was drawn on cycle day 9 and analyzed for the percentage of circulating T cells expressing the activation markers CD69+ and human leukocyte antigen (HLA)-DR and the suppressor marker CD11b using immunofluorescence and flow cytometry. These results were compared with total percentage circulating CD3, CD4 and CD8 cells as well as NK cells and pregnancy outcome that cycle. Results: Infertile women had significantly greater expression of the activation marker of CD69+ among CD8+ and CD4+ T cells and HLA-DR among CD4 cells than fertile women. No difference in expression of T cell suppressor marker of CD11b was noted when infertile and fertile women were compared. No correlations were observed when activated T cells were compared with circulating CD3+, CD4+, CD8+, activated NK cells and NK cytotoxicity. CD3+4+HLA-DR+ was expressed significantly less among successfully pregnant compared with unsuccessfully pregnant women. Conclusion: T-cell activation markers CD 69+ and HLA-DR+ are associated with increased implantation failure after IVF/ET.

Purpose: The pivotal event in determining successful from unsuccessful cycles after in vitro fertilization is implantation. The purpose of this study was to compare the percentage of circulating NK cells expressing activation and inhibition markers between infertile and fertile control women and to determine the correlation between these markers and those of the NK cytotoxicity activation assay. Lastly, we wish to determine the ability of each of these markers to predict pregnancy outcome after IVF/ET (in vitro fertilization/embryo transfer). Methods: Blood samples from 22 infertile women undergoing IVF/ET during the November 2001 cycle were drawn on cycle Day 9 and analyzed for expression of CD69+, HLA-DR, CD161+, CD94+, and CD158a+ as well as NK cytotoxicity using immunoflluorescent labeling and flow cytometry. Results were compared with those from 26 fertile control women and correlated to pregnancy outcome that of cycle. Results: Infertile women had significantly higher expression of NK cell activation markers of CD69+ and CD161+ than fertile women. NK cytotoxicity correlated inversely with expression of NK cells bearing the inhibition marker of CD94+. None of the successfully pregnant women of that cycle had elevated levels of NK cytotoxicity whereas 50% of those experiencing a chemical pregnancy loss and those not becoming pregnant had elevated levels of NK cytotoxicity. Conclusions: Immunologic markers can identify mechanisms involved in implantation failure. Activation markers of CD69+ and CD161+ expressed on NK cells as well as NK cytotoxicity can be added to the previously reported risk factors for immunologic implantation failure.

Coulam CB, Roussev R. Chemical pregnancies: immunologic and ultrasonographic studies. AJRI 2002; 48:323–328 © Blackwell Munksgaard, 2002 PROBLEM: Implantation of the embryo determines successful from unsuccessful cycles after in vitro fertilization (IVF) and embryo transfer (ET). The purpose of this study was to compare immunologic risk factors among women experiencing implanation failure characterized by a negative pregnancy test after IVF/ET and those experiencing chemical pregnancies. In addition ultrasonographic measurement of gestational sac size from 24 to 35 days from last menstrual period (LMP) were compared between chemical pregnancies and other pregnancy outcomes. METHODS OF STUDY: Blood samples from 122 women experiencing IVF implantation failure with a negative pregnancy test after ET and 20 women with chemical pregnancies were evaluated for the presence of antiphospholipid antibodies (APA), antinuclear antibodies (ANA), circulating embryotoxins (ETA) and elevated levels of natural killer (NK) cells. Gestational sac size measured from 24 to 35 days form LMP were compared according to pregnancy outcome: term birth (n=46), ectopic pregnancy (n=49), spontaneous abortion (n=56) and chemical pregnancy (n=20). RESULTS: Women experiencing chemical pregnancies had a higher frequency of APA than women with implantation failure associated with a negative pregnancy test (80% versus 28%, P

Problem A recent hypothesis has implicated superfertility as a cause of recurrent pregnancy loss. Clinical support for the concept comes from one report that 40% of women experiencing recurrent miscarriages had monthly fecundity rates of 60% or greater and thus were designated as superfertile. Methods of study To confirm or refute this finding, clinical histories of 201 women with a history of recurrent pregnancy loss were reviewed and months to desired pregnancy, karyotypes of their products of conception as well as results of laboratory tests including antiphospholipid antibodies and circulating natural killer cells were recorded. Results The prevalence of superfertility was 32% (64/201) among recurrently aborting women compared with 3% of the general population according to the model of Tietze (P

The prevalences of antiphospholipid antibodies (APAs) among 1,325 women with a history of unexplained infertility and 676 women experiencing recurrent implantation failure were compared with 789 women experiencing recurrent pregnancy loss and 205 fertile control women. Eight percent and 9% of women with a history of unexplained infertility and recurrent implantation failure had more than one positive APA compared with 1.5% of fertile negative control women and 11% of positive control women experiencing recurrent pregnancy loss.

Premature menopause and unexplained infertility are associated with ovarian antibodies, a marker of ovarian autoimmunity. In premature menopause, FSH is also elevated while in unexplained infertility FSH concentrations are often normal. The relationship of ovarian antibodies and FSH and inhibin B, as markers of follicle function, was investigated in unexplained infertility. Ovarian antibodies were determined by immunoassay in comparison to normal controls (n = 12); 51.9% were positive at two SD (P0.05) and 38.5% were positive at three SD above the control mean (P0.01). In this study three SD above the control mean was considered positive. In unexplained infertility, three out of 10 (30%) had elevated day 3 FSH (10 mIU/ml) and ovarian antibodies, while 17/42 (40%) had normal FSH (10 mIU/ml) and ovarian antibodies. In women with normal FSH, two out of seven (29%) had low inhibin B concentrations (33 pg/ml) and ovarian antibodies, and 15/35 (43%) had normal inhibin B concentrations (33 pg/ml) and ovarian antibodies. Similarly, when women with and without ovarian antibodies were compared there was no difference in mean FSH or mean inhibin B concentrations. Thus, unlike other endocrine autoimmune disorders, hormone concentrations are not predictors of potential ovarian autoimmunity. This suggests that in unexplained infertility ovarian antibodies are an independent marker of potential ovarian failure, and may precede changes in regulatory hormones.

The use of egg yolk and serum albumin as additive diluents for human sperm cryopreservation is routine. But because both diluents are of animal origin, they potentially may introduce microbial agents to the sample. To reduce the risk of contamination, the cryoprotective property of phospholipids extracted from lecithin was evaluated and found to be effective when supplemented with dimethyl sulfoxide and glycerol.

Purpose:To evaluate the contribution of embryo quality to preclinical loss rates after in vitro fertilization (IVF)/embryo transfer (ET) pregnancy, multiple gestation, and clinical loss rates were compared to preclinical pregnancy loss rates over a 3-year period.

Are Antimullerian Hormone and its Receptor Genes Associated with Low Ovarian Response? One of the most frustrating problems in the treatment of infertility is that of poor ovarian response to stimulation. It therefore, would be advantageous to have a genetic marker that could predict low ovarian reserve at a young age before the impact of low ovarian reserve affects a woman’s fertility. Antimullerian hormone (AMH) has emerged as the the most accurate measure of ovarian reserve. The purpose of the present study is to investigate the correlation between AMH and AMH Receptor II (AMHRII) polymorphisms and low ovarian reserve.

Recurrent spontaneous abortion is a health care concern occurring in 2-5% of couples' attempting to reproduce. The observed frequency of recurrent abortion is higher than that expected by chance (0.3%), which implies an underlying cause(s) contributing to the recurrent Effective treatment is needed for couples experiencing recurrent spontaneous abortion. Before effective treatment can be instituted, the cause of pregnancy loss must be determined. The underlying causes of recurrent spontaneous abortion are not entirely clear. As a result, controversy regarding the appropriate evaluation and treatment of couples experiencing recurrent spontaneous abortion ex'ists. To approach this controversy the American Society for Reproductive Immunology, under the direction of its President, Thomas J Gill, 111, established the committee on Clinical Guidelines Recommendation Committee for Recurrent Spontaneous Abortion in 1995. The members of the Committee were: Carolyn Coulam, Chairman; Alan Beer; William H. Kutteh; David A Clark; Robert Silver; Joanne Kwak; and Mary Stephenson. Each member was asked to submit guidelines for both diagnosis and treatment of recurrent spontaneous abortion. The guidelines were collated by the chairman and circulated to the members of the committee for additions and corrections. A final corrected report was recirculated to the committee members, and the committee met at the time of the 16th Annual Meeting of the American Society of Reproductive Immunology on June 23, 1996, in Knoxville, Tennessee, for a consensus conference. The final manuscript emerging from the consensus conference was given to the Council of the American Society for Reproductive Immunology for their consideration. Additions and corrections made by the council were incorporated into the report, and the report was recirculated to the new council and committee members before the Interim Executive Council Meeting on December 7, 1996, in Pittsburgh, Pennsylvania. The Committee presents options for diagnosis and treatment of recurrent spontaneous abortion. The Clinical Options for Diagnosis of Recurrent Spontaneous Abortion are based on an evaluation determined by risk factors of recent experimental and empirical research. Risk factors for recurrent spontaneous abortion in

Purpose: Our purpose was to determine the specific anti-phospholipid antibodies (APAs) that should be evaluated to identify individuals at risk for implantation failure associated with reproductive autoimmune failure syndrome (RAFS).

Objective To compare outcome of pregnancies after intracytoplasmic sperm injection (ICSI) with those of other assisted reproductive technologies. Design Pregnancy outcomes after ICSI were followed prospectively and compared with pregnancy outcomes after IVF with fresh and frozen ETs and donor oocyte cycles. Setting A private tertiary referral center for genetics and infertility in Fairfax, Virginia. Patients One hundred thirty-six couples achieving pregnancy after undergoing ICSI, 71 after IVF, 35 donor oocyte recipients, and 19 after transfer of frozen-thawed embryos. Interventions In vitro fertilization and/or ET for all couples. Dilatation and curettage to obtain products of conception for chromosome analysis in 28 women experiencing spontaneous abortion. Main Outcome Measures Pregnancy outcomes were classified as preclinical loss, clinical loss, and ongoing pregnancy. Results The mean frequency of preclinical pregnancy loss was 26% after ICSI, 28% after IVF, 3% after ET using donor oocytes, and 11% after frozen ET. The rate of clinical loss after ICSI (21%) was compared with IVF (18%), donor oocyte cycles (11%), and frozen ETs (21%). Conclusions Intracytoplasmic sperm injection is not associated with an increase in pregnancy losses, clinical or preclinical, compared with conventional IVF.

PROBLEM : Tests to determine presence of embryos prior to implantation are needed. METHODS : Sera from women after embryo transfer were tested for preimplantation factor (PIF) using the lymphocyte/platelet binding assay. Autorosettes were counted using blood type O+ donor lymphocytes and platelets incubated with blinded serum in the presence of antiCD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte/platelet assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The roles of platelet activating factor (PAF) and chaperonin 10 in the observed phenomena were studied experimentally. RESULTS : Significantly more lymphocyte/platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. Neither PAF nor chaparonin added to the tested sera controls influenced the percentage of lymphocyte/platelets rosettes. CONCLUSIONS : PIF is a likely candidate to be the next frontier of diagnosing the presence of viable preimplantation embryos in vivo.

Preimplantation factor (PIF) is detected in the serum of women shortly after fertilization; its origin, however, has not been established. In this study, the embryonal origin of PIF was investigated and partial characterization of the factor was carried out. Culture media from viable human 2-8-cell stage embryos and mouse 2-cell-blastocyst stage embryos were analysed using the lymphocyte/platelet binding assay (LPBA). The assay was performed by combining culture media with donor O+ type blood-derived lymphocytes/platelets, complement and an antibody against CD2. Increased autorosette formation between lymphocytes and platelets (> 9%) was an indication for the presence of PIF. In addition, the effect of platelet-activating factor (PAF) and chaperonin 10 on PIF activity was determined. Partial purification of PIF was carried out using gel filtration and reverse-phase high purification liquid chromatography (HPLC), followed by mass spectrometry. Culture media of single human viable fertilized oocytes were negative for PIF; however, the 10-fold concentrated medium was positive for PIF. In medium in which five or more mouse embryos were cultured, PIF activity was observed starting at the morula stage and was higher by the blastocyst stage. Addition of PAF or chaperonin 10 to the PIF assay did not elicit a specific effect on PIF activity. Chromatographic data suggest that PIF activity is due to low molecular weight proteins. PIF appears to be a low molecular weight protein which is derived from viable preimplantation embryos. It is different from PAF or chaperonin 10. Its final characterization will be valuable for better understanding of maternal recognition of pregnancy and implantation.

PROBLEM: Efficacy of immunotherapy for treatment of recurrent spontaneous abortion (SA) has been controversial. The low treatment effect of white blood cell immunization lead to investigations of alternative treatments including intravenous (IV) immunoglobulin (Ig). To evaluate the efficacy of IVIg for treatment of recurrent SA, a prospective, randomized, double blinded, placebo-controlled trial was performed. METHODS: Ninety-five women experiencing two or more consecutive spontaneous abortions with no known cause were randomized and received either IVIg 500 mg/kg/month or placebo (albumin). RESULTS: Of 95 women participating in the study, 47 received IVIg and 48 received placebo. Medication was discontinued in 34 women who failed to conceive within four cycles. The remaining 61 women achieved pregnancy. Pregnancy outcomes included 29 deliveries and 32 recurrent SA. Among women delivering live births 18 (62%) received IVIg and 11 (38%) received placebo. By contrast, 21 (66%) women experiencing recurrent SAs received placebo and 11 (34%) received IVIg. Among 61 women who conceived, 29 received IVIg and 32 received placebo. Of the 29 women who conceived and received IVIg, 18 (62%) delivered live births and 11 (38%) experienced recurrent SA. Of 32 women who conceived and received placebo 11 (34%) delivered live births and 21 (66%) had recurrent SA. The difference in live birth rates between women receiving IVIg and placebo was significant (P = 0.04, odds ratio 3.1). CONCLUSION: IVIg is effective in enhancing the percentage of live births among women experiencing unexplained recurrent SA.

PROBLEM: The presence of embryotoxic factors in sera from women with recurrent spontaneous abortion (RSA) has been proposed as a basis for classification of unexplained RSA. To determine the prevalence of circulating embryotoxins among women with idiopathic RSA, sera from 160 women were studied using the mouse blastocyst assay. METHODS: Two-cell embryos were collected from superovulated mated CB6F1/J mice and cultured in media supplemented with fetal bovine serum (FBS) or 10% serum at 37°C with 5% CO2 and high humidity. Each assay was run in triplicate using three mice with at least five embryos from each mouse. Results were determined by calculating the average percentage atresia for each mouse. FBS, known to support embryo proliferation, was used to control in each assay. RESULTS: The prevalence of embryotoxic factors among women experiencing RSA was 24.4% (39/160). There is no correlation found between the presence of embryotoxicity and phospholipid antibodies, lupus anticoagulant, and thyroglobulin/microsomal antibodies. CONCLUSION: The embryotoxicity assay can serve as a basis for a new approach for classification of unexplained recurrent spontaneous abortion.

PROBLEM : TJ6 is a cytokine whose membrane form is regularly expressed on the B lymphocytes of women during pregnancy. In a previous study we have shown that pregnancies that end in a spontaneous abortion are characterized by an increase in natural killer (NK) cells and that NK cells in these pregnancies also expressed TJ6, whereas NK cells from pregnancies that terminate normally neither increase in number nor do they express significant amounts of the cytokine. METHODS : To evaluate the ability of TJ6 to predict pregnancy outcome, 61 blood samples from pregnant women were studied. Blood samples were drawn between 5 and 12 weeks of gestation and analyzed for lymphocyte expression of TJ6 using Cytoron Absolute flow cytometry and two-color fluorescence. The percentage of CD19+ (B) cells and CD56+ (NK) cells that expressed TJ6 was calculated, and this percentage was correlated with subsequent pregnancy outcome classified as successful (viable birth) or unsuccessful (abortion, still-birth). TJ6 CD19 and TJ6 CD56 was determined in 32 blood samples from women with successful pregnancy outcomes and 29 samples from women with unsuccessful pregnancy outcome. RESULTS : The mean TJ6 CD19 expression for successful pregnancies was 10.6% and for unsuccessful pregnancies was 5.1% (P < 0.03). The mean TJ6 CD56+ percentage of circulating cells that express TJ6 expression for successful pregnancies was 3.3% and for unsuccessful pregnancies was 10.4% (P = 0.02). All unsuccessful pregnancies had less than 90% of circulating CD19 cells express TJ6 and/or greater than 50% of circulating CD56 cells express TJ6. Use of the expression of TJ6 on CD19 and CD56 cells during the first trimester predicts viable pregnancy with a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 100%. CONCLUSIONS : The work now being reported validates the hypothesis that expression of TJ6 on NK cells obtained from pregnant women predicts the outcome of pregnancy. In addition we show that this prognosticator can be reliably demonstrated early in the first trimester.

Objective To evaluate, in a prospective study, the fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) in infertile couples with severe male infertility. Design Intracytoplasmic sperm injection was performed in 229 consecutive IVF cycles on 190 couples with rigorously defined severe male infertility or proven failure of fertilization in prior IVF cycles. Neither male nor female partners were chosen from a waiting list or on any other selective basis, including age, prior or anticipated ovarian response, or oocyte number or quality. There were no upper age limits, in no instance was donor sperm used for ICSI, and cycle cancellation rate was minimal. Setting Private genetics and fertility center in Fairfax, Virginia. Main Outcome Measures Fertilization, transfer, and pregnancy rates were measured in ICSI-treated couples, and comparisons were made regarding both female age and strictly defined semen categories. Results Two hundred six cycles (90%) resulted in ETs, with initiation of 52 pregnancies (25% per transfer, 23% per cycle). Thirty-eight of 52 (18% per transfer) were clinical pregnancies with established gestational sacs or were ongoing or delivered. Pregnancies were achieved even in older women but were more readily established in younger women producing larger numbers of metaphase II oocytes. The severity of semen abnormalities had some small effect on fertilization rate, but only actual necrospermia was associated with markedly decreased frequency of embryo formation. Pregnancy per transfer was similar across groups. In some cases, pregnancy was initiated with fewer than 100 viable sperm in the ejaculate. Conclusions Intracytoplasmic sperm injection is a very powerful new treatment for severe male infertility. Paradoxically, egg number and probably egg quality are now the main determinants of success in treating male infertility.

PROBLEM: Compare data from several centers relating to success rates in recurrent spontaneous miscarriage and assess the significance of indicators of subsequent pregnancy loss. METHOD: Data from 777 couples with unexplained recurrent spontaneous abortion from independent studies at seven centers were analyzed using logistic regression analysis. The following covariates were considered: age of patient, number of previous spontaneous abortions, length of previous abortions history, sub-fertility index (defined as the product of the number of spontaneous abortions and the abortion history), whether a patient was a primary or secondary aborter, and whether a patient had received leukocyte immunotherapy. RESULTS: There was a highly significant difference between the seven centers in success rates in the subsequent pregnancy and a highly significant association between success rate and each of the following covariates: the number of previous abortions, the length of the previous abortion history and the sub-fertility index. In particular, for each increase of 10 units in the value of the sub-fertility index, up to a value of 30, the odds in favor of a successful pregnancy decreased by a factor of 0.6, i.e., 40%. There was, however, little evidence of an association between the success rate in the subsequent pregnancy and age, parity, or immunization with cells from the husband. CONCLUSIONS: The sub-fertility index may be a useful measure of likelihood of success in a subsequent pregnancy.

PROBLEM: To evaluate differences in circulating CD56+ cells between successful and unsuccessful pregnancies, 114 pregnant women were studied prospectively. METHOD: Seventy women had a history of infertility (INF) and 44 had two or more previous spontaneous abortions (RSA). Among the infertile women, 12 were donor egg recipients (DER) and 15 underwent intracytoplasmic sperm injection (ICSI) for treatment of male factor infertility. Nineteen women were varrying multiple gestations (MG) and 55 had singleton gestations (SG). Thirteen additional women were receiving intravenous immunoglobulin (IVIg). RESULTS: The percentage of CD56+ cells was determined in 310 blood samples from 114 pregnant women by flow cytometry. The prevalence of women with persistent elevation of percent of 56+ cells (>12%) was 58% among DER, 73% among ICSI, 37% among MG, 22% among SG, 18% among RSA, and 39% among INF. Thirteen women with SG received IVIG, 10 had CD56+ cells greater than 12% and all 13 experienced live births. Women with percentage CD56+ cells persistently greater than 12% who were not DER, not ICSI, not receiving IVIg, and not varrying MG had a live birth rate of 11%. Women with greater than 12% CD56+ cells had normal karyotype in 78% of concepti studied in contrast to women less than 12% CD56+ cells who had 68% abnormal karyotypes (P=0.04). CONCLUSION: Elevated CD56+ cells in pregnant women who are not DER, not ICSI, not receiving IVIg, and not carrying MG predicts loss of a karyotypically normal conceptus with a specificity of 87% and positive predictive value of 78%. While the specificity value of this test is high in both infertile and RSA populations, the sensitivity is 86% in RSA and only 54% in INF suggesting this test does not identify all losses among INF. It may identify a subset of pregnancies at risk for loss of a karyotypically normal embryo that may respond to treatment with IVIg

Before effective treatment for reproductive failure can be instituted, the cause of the failure must be determined. A search of PubMed was made to identify the published data regarding diagnosis and treatment of reproductive failure. Results were compared with the frequency of antiphospholipid antibodies (APA) in 2995 women with histories of unexplained infertility, recurrent implantation failure, recurrent pregnancy loss, and fertile women. In addition, pregnancy outcomes among 442 women experiencing reproductive failure and elevated NK cell activity after treatment with intravenous immunoglobulin (IVIg) (N = 242) or intralipids (N = 200) were compared. The prevalence of APA was the same among women with the diagnosis of unexplained infertility, recurrent implantation failure, and recurrent miscarriage. Heparin and aspirin are successful in the treatment of elevated APA among women with recurrent miscarriage but not with recurrent implantation failure. IVIg has been successful in the treatment of recurrent miscarriage and recurrent implantation failure among women with elevated APA and/or NK cell activity. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cell cytotoxicity treated with intralipid were compared with women treated with IVIg, no differences were seen. Immunotherapy for treatment of reproductive failure enhances live birth but only in those women displaying abnormal immunologic risk factors.

PROBLEM: Recurrent spontaneous abortion (RSA) is the cause of childlessness in 2–5% of reproducing couples. Immunological mechanisms have been proposed as an etiology in some cases of RSA. Various forms of immunotherapy have been attempted in individuals thought to have an immunologic mechanism associated with RSA. Intravenous immunoglobulin (IVIG) has been used in a pilot study to successfully treat women with RSA. METHOD: To evaluate the efficacy of IVIG in the prevention of RSA as prospective randomized, placebo-controlled clinical trial was undertaken. Women experiencing two or more consecutive RSAs receive either IVIG 500 mg/kg/month or placebo (albumin). RESULTS: To date 92 women have been enrolled in the study and 58 pregnancies have been achieved. The outcome of the 58 pregnancies include 20 deliveries, 9 ongoing pregnancies and 29 losses. Fourteen (49%) of the 29 pregnancy losses were blighted ova (empty gestational sacs) and 15 (51%) were intrauterine fetal deaths (IUFD's) occurring in the first trimester of pregnancy. Of 14 blighted ova, 8 were in women receiving IVIG and 6 were receiving placebo. Fifteen IUFD's occurred: 3 (20%) in women receiving IVIG and 12 (80%) placebo. Of 11 pregnancy losses occurring in women receiving IVIG, 8 (73%) were blighted ova and 3 (27%) were IUFD's. Eighteen losses occurred in women receiving placebo: 6 (33%) were blighted ova and 12 (67%) were IUFD's. CONCLUSIONS: These preliminary data suggest IVIG may be effective treatment for RSA. Analysis of data from the completed randomized placebo-controlled trial will test this suggestion.

The aim of this study was to determine the effectiveness of intravenous (i.v.) immunoglobulin (Ig) for treatment of individuals experiencing failure after in-vitro fertilization (IVF) and embryo transfer. A total of 29 women with unexplained infertility who failed to become pregnant after IVF/embryo transfer were divided into two groups based on performance in previous IVF cycles: 16 women had fertilization of > or = 50% of oocytes retrieved and/or produced > or = 3 embryos each cycle and 13 had fertilization of or = 50% and generate at least three embryos per cycle.

PROBLEM: Recurrent spontaneous abortion (RSA) is a common complication of pregnancy for which there is no known cure. Therefore, effective treatment is needed. Published results from controlled clinical trials of allogeneic leukocyte immunization of women suffering from RSA have given conflicting results. To address this controversy, the international raw data of all patients who had been entered into clinical trials that included a control group were collected and analyzed. The primary question to be answered was whether alloimmune stimulation of the female partner improves the subsequent live birth rate. METHODS: Fifteen clinical centers were identified worldwide because they controlled appropriate raw data. Consequently, nine randomized trials (seven double-blinded) were evaluated independently by two separate data analysis teams to assure conclusions were robust. One team also compared randomized trials to the results of six nonrandomized cohort-controlled studies to test for bias in nonrandomized trials. Factors predicting successful live births among couples with RSA were evaluated by logistic regression. RESULTS: Although the two independent analyses made use of different definitions and utilized different statistical methods, the results of both were similar. The live birth ratios (ratio of live births in treatment and control groups) with 95% confidence intervals (CI) were 1.16 (CI, 1.01-1.34, P = 0.031) and 1.21 (CI, 1.04-1.37, P = 0.024), respectively. The absolute differences in live birth rates between treatment and control groups were 8% and 10% in respective analyses. Results in randomized and nonrandomized trials were surprisingly similar despite significant differences in composition of control and treatment groups. Live birth rates were lower with older female partners, more than five abortions, with a positive ANA or with positive anticardiolipin antibodies. Live birth rates were higher if the female partner had prior to treatment serum antibodies to paternal leukocytes or converted from negative to positive with immunization. Approximately 0.5% of controls and 2.1% of treated patients experience side effects for a 1.6% treatment related effect. There was no evidence of an increased risk of adverse effects on the fetus. CONCLUSIONS: Two independent analyses of worldwide data on allogeneic leukocyte immunization for treatment of RSA suggest that alloimmunization may be an effective treatment. The treatment effect appears, however, to be small, and the data indicate that immunotherapy helps only 8% to 10% of affected couples. A current lack of diagnostic tests defining patients who most likely would benefit from immunotherapy, precludes the identification of a patient population that would benefit most from such treatment. The efficacy of treatment in such a subgroup could be expected to increase and could be of sufficient magnitude to allow the determination of more effective immunization protocols. This study does not exclude the possibility of a partial correction of a widely prevalent immunology defect by immunotherapy. The presence of such a defect would indicate a need for more effective therapy. The unexplained variation in pregnancy success rates of control groups among centers continues to present a statistical problem, limiting the statistical evaluation of retroactively obtained data.

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients (n=14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14-3.55, chi-squared=12.74, P=0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11-0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared=4.0, P=0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored.

To evaluate the usefulness of high-resolution ultrasound with colour Doppler mapping system in performing transcervical wire tuboplasty, 13 infertile women with bilateral proximal tubal obstruction demonstrated by X-ray or colour Doppler ultrasound hysterosalpingography and chromoperturbation at laparoscopy were treated by transcervical wire tuboplasty utilizing colour Doppler mapping ultrasound guidance. A co-axial catheter was used to introduce a 0.016-in. flexible wire to the occluded portion. Transcervical wire catheterization was accomplished in 25 of 26 tubes (96%), resulting in patency in 24 of 25 tubes (96%). At the end of the procedure all patients had at least one tube patent (100%). Five (38%) women achieved pregnancies within 1 year after the procedure. High-resolution ultrasound with colour Doppler mapping system is an efficacious way to perform transcervical wire tuboplasty, avoiding the risk of radiation and allergic reaction.