Diagnostic Tests for Reproductive Medicine

The following tests are very specific and should be used by professionals or very well aware couples that suffer from infertility issues, RSAs, or failed IVFs.

The first issue that a couple has to account when searching what is wrong with them and don’t have a baby yet is to exclude that the husband has a sperm problem. Besides the normal sperm count, motility etc. performed to most diagnostic tests a following test should be accounted for all men that want to know whether they are potential and fertile.


 

DNA Fragmentation or DFI

Results of recent research indicate that sperm quality influences not only rates of fertilization of eggs, but also subsequent embryo development. In humans, these paternal effects have been shown to affect rates of embryo cleavage, blastocyst formation and implantation. The markers of sperm quality used to predict pregnancy outcome are not the parameters included in the standard semen analysis (sperm concentration, motility or morphology), but rather the results of the Sperm DNA fragmentation assay or DFI. This specific test measures DNA damage in sperm and is reported as DNA fragmentation index (DFI) and high DNA staining (HDS). The mechanism by which damaged sperm DNA affects pregnancy outcome is not fully known.

Indication for Testing

Male partners of couples with a history of unexplained infertility, poor embryo quality after in vitro fertilization (IVF), implantation failure after IVF, recurrent chemical or occult pregnancy losses, or recurrent early spontaneous abortions should be tested with Sperm DNA fragmentation assay. The myth that fertility was only women’s issue is false because the new statistics show that for infertility issues nowadays the rate of 50% belongs to men that have sperm issues.

Detection Method

Sperm DNA fragmentation assay is based on utilization of metachromatic features of acridine orange , a DNA probe, and the principles of flow cytometry. The assay measures the susceptibility of DNA to denaturation in situ after low pH treatment what denatures protamine associated DNA in sperm cells but does not denature DNA associated with histones. Spermatozoa with normal chromatin structure do not demonstrate DNA denaturation. Acridine orange that intercalates into dsDNA (normal) fluoresces green, while acridine orange that associates with ssDNA (denatured) fluoresces red when excited by 488nm light source.

Specimen Requirements:

A semen sample should be collected by masturbation and allowed to liquefy at room temperature for approximately 30 minutes. The semen specimen should be transferred directly into liquid nitrogen (or -70oC freezer) in a nunc tube labeled with the patient’s name and date. Semen can be kept for up to 5 hours at room temperature prior to freezing without loss of quality. The frozen samples should be shipped overnight in liquid nitrogen (or dry ice containers).

Range or Results:

High fertility potential < 15%, Fair fertility potential 15%-30%, Poor fertility potential> 30%

Therapy: International scientific team consults couples and gives the best targeted individualized treatment for any sperm issues that account for DNA fragmentation test.


 

Women’s Tests

Because the woman’s reproductive system is more complicated and more peculiar many more tests have been developed for her. The regular tests such as testing of hormones and all microbiology markers should be tested in case of infections. However, the most important part offered by our international labs is the specific immune tests that account for more than 80% for reproductive issues.  All tests can be seen in the test request form provided here:

Female diagnostic for ovary reserve and quality: inhibin B and AMH.

INHIBIN B

Inhibin-B serum concentration provides a new measure of ovarian reserve. Ovarian reserve describes the ovary’s capacity to respond to gonadotropin stimulation by producing a sufficient number of good quality eggs capable of generating normal embryos. Granulosa cells of the ovarian follicle secrete Inhibin-B. Most of the serum Inhibin-B concentration originates from large or dominant follicles since these follicles secrete ten fold higher concentrations follicles measuring 4mm. Inhibin-B controls FSH secretion form the pituitary gland. Thus, Inhibin-B is a more direct measurement of assessing ovarian function than FSH. Inhibin-B serum concentrations drawn on cycle day 3 have been shown to predict response of ovaries to gonadotropin stimulation in in vitro fertilization (IVF) cycles. Women who had less than 45pg/ml Inhibin-B on cycle day 3, required 50% more ampules of the day of hCG, 33% reduction in the number of oocytes retrieved, less embryos transferred per cycle and 70% reduction in pregnancy rate, than women with day 3 Inhibin levels greater than 45pg/ml. The women with day 3 Inhibin levels less than 45pg/ ml that did get pregnant had an 11 fold increase in spontaneous abortions compared with greater than 45pg/ml.

In fertile males inhibin-B is produced by the Sertoli cells (the site of the sperm production) and the level of it can accurately predicts a viable sperm. Inhibin-B has been reported to predict the outcome of TESE and ICSI among men with non-obstructive azospermia. Sperm were significant less likely to be recovered from men with low Inhibin-B concentration.

Indications for Testing

Women aged 35 years or greater, women of any age experiencing unexplained infertility who have a poor response to ovulation induction, women presenting for other tests of ovarian reserve including day 3 FSH and clomiphene challenge test, and women experiencing recurrent pregnancy losses should be tested for day 3 Inhibin B concentrations. All azospermic men undergoing TESA for ICSI and IVF, can be offered  the inhibin-B test.

Detection Method

Inhibin B is detected by standard ELISA procedure


 

AMH (anti-Mullerian Hormone)

Anti-Müllerian hormone (AMH) or Müllerian-inhibiting substance is known to be instrumental in the determination of gender in the embryo, inducing testicular differentiation in the male and permitting development of the female reproductive tract in its absence. However in the cycling female, AMH is secreted by preantral and small antral follicles in the ovary, and circulating levels positively correlate with the number of remaining ovarian follicles. Thus, AMH levels reflect ovarian reserve (ie, the number of primordial follicles present). Indeed, AMH levels positively correlate with the antral follicle count commonly used as a measure of ovarian reserve.

It can help clinically to determine ovarian reserve in cycling women (AMH has been suggested as the best biochemical marker of ovarian reserve, the higher the AMH level the higher the number of remaining follicles), to predict likelihood of success and avoid hyperstimulation syndrome in women undergoing in vitro fertilization (IVF), and to diagnose polycystic ovary syndrome.

Detection Method

Anti-Müllerian hormone is measured using dual monoclonal antibodies in a chemiluminescent immunoassay (CLIA). No significant cross-reactivities were identified. Analytical sensitivity (LoQ) is 0.03 ng/mL.

NOTE: For this important part of these 2 diagnostic tests for woman;s quality of ovaries, specialized scientific team can evaluate and can consult for special treatment before menopause.


 

Immune System Tests

Reproductive Immunophenotype

Introduction

Identification of the type of relative concentrations of various white blood cell populations in blood is valuable in determining risk factors for pregnancy loss. The Reproductive immunophenotype has been shown to be useful in identifying individuals at risk for not implanting embryos and for loosing karyotypically normal pregnancies due to elevated circulating Natural Killer (CD56+) cells. The Activated Reproductive Immunophenotype measures not only the percentage of circulating lymphocytes as the Reproductive Immunophenotype does, but also activated NK and T cells.  Women experiencing implantation failure after IVF/ET have significantly higher expression of NK cell activation marker of CD69+ and of T cell activation marker of HLA-DR.

Purpose

All women experiencing two or more consecutive pregnancy losses or who fail to conceive after in vitro fertilization and embryo transfer should be tested for the presence of elevated percentage of CD56+ cells.  Elevated percentage of CD56+ cells should be monitored during pregnancy to aid in treatment management.

Detection Method

The percentages of mononuclear cells expressing CD3, CD4, CD8, CD19, CD16, and CD56 as well as CD56+16+69+, CD56+16-69+ and  CD3+4+DR+ are detected by flow cytometry using an immunofluoroescence technique.

Specimen collection

20ml of whole blood collected in two 10ml sodium heparin (green-topped) tubes.  Ship at room temperature.


 

NK (Natural Killer Cell) ACTIVATION ASSAY

Introduction

Natural Killer (NK) cells are a lymphocyte subset that express CD56+ on their cell surface. They are large granular lymphocytes that arise from the bone marrow and circulate peripherally before localizing to specific tissues. NK cells are the most prevalent lymphocyte population in secretory endometrium and in decidua of early pregnancy. They are believed to play a role in regulating trophoblastic cell migration. The NK cell activation assay tests the killing function of circulating NK cells as well as the ability of interleukin-2 (IL2) to stimulate and intravenous immunoglobulin (IVIg) to suppress that activity. NK activity can be measured as a bioassay that determines the ability of activated NK cells to kill their target (K562 cell line).

Increased peripheral and endometrial NK killing activity have been demonstrated among patients undergoing in vitro fertilization (IVF) who experienced spontaneous pregnancy loss or IVF failure. Interestingly, 40% of patients undergoing IVF with a diagnosis of endometriosis displayed increased NK activity. Identification of patients with increased NK cell activity will allow appropriate treatment for those who will benefit from it.

Indications for Testing

All women who have unexplained infertility, endometriosis or other pelvic pathology such as pelvic adhesions, a history of recurrent IVF failures, have elevated APA or ATA and are undergoing an IVF procedure, or a history of two or more consecutive pregnancy losses should be tested with the NK Cell Activation Assay (NKa).

Detection Method

The percentage of lysed K562 cells is detected by flow cytometry using an immunofluorescence technique.

Specimen Requirements

Collect one (2) 6ml green top tube. Ship at room temperature overnight, next day morning. For longer destination a special tube would be advised that can keep lymphocytes for 10 days stable.


 

ETA (EMBRYOTOXICITY ASSAY)

Introduction

Recurrent pregnancy loss, unexplained infertility, and infertility associated with endometriosis affect a large number of reproductive age couples. Data are accumulating to suggest that a proportion of these individuals have circulating embryotoxins associated with their reproductive failure. It has been proposed that positive and negative embryotoxic factors provide the basis for a new classification of idiopathic recurrent pregnancy loss and in vitro fertilization (IVF) failure. The prevalence of embryotoxic activity among women experiencing recurrent pregnancy loss is 10% to 15% and among IVF failures is 20% to 25%. Embryotoxic factors have also been reported in women experiencing unexplained infertility and infertility associated with endometriosis.

Indication for Testing

All women experiencing unexplained infertility, implantation failure after IVF, infertility associated with endometriosis or two or more consecutive pregnancy losses should be tested for circulating embryotoxins.

Detection Method

Embryotoxic factors are identified by screening the patient’s serum by using mouse embryos.

Specimen Requirements:

Collect one (1) 8 ml red or tiger top tube. Centrifuge and get the serum. Then ship at room temperature overnight, next day morning.


 

BLOCKING FACTORS

It has been established that normal pregnancy requires the presence of blocking factors (antibodies) in woman’s blood circulation. Such factors are not detected in most women with recurrent pregnancy losses (RPL) or unexplained imminent abortions (IA) or failed IVFs.

Detection Method

Lymphocytes from previously HLA-phenotyped donor have been used. The lymphocytes were incubated with one specific anti-HLA antibody and used ex-tempore or stored frozen in liquid nitrogen until used. The lymphocytes with HLA-Ag-Ab complex were incubated with the tested serum for 30 min. A rabbit complement was added and after an hour of incubation, a coloring agent (eosin or tripan blue) is dropped. The results are scored on light microscope: if blocking factors are present in the serum, no cellular lyses are detected and vice versa – blocking factor’s absence will lead to cell lyses.

Result

Blocking Factors Result  

Presence of blocking factors

Weak positive (+

positive (+)

Specimen Collection

Lymphocytes are needed for husband so 1 green top necessary and serum from wife.


 

ANTIPHOSPHOLIPID ANTIBODIES (APA)

Introduction

Antiphospholipid Antibodies (APA) are a group of organ nonspecific autoantibodies that bind to both anionic and zwitterionic phospholipids. Their presence has been associated with reproductive failure characterized clinically as recurrent pregnancy loss, unexplained infertility and infertility associated with endometriosis. Anti phospholipid antibodies have also been associated with thrombosis. The most consistently reported phenomenon is the association between recurrent pregnancy loss and the presence of APA; and treatment of APA positive women with heparin or prednisone and aspirin or intravenous immunoglobuhn improves pregnancy outcome among these women in all studies published.Women who present for in vitro fertilization (IVF) have been consistently shown to have a high prevalence of APA, especially those with pelvic pathology such as endometriosis and pelvic adhesions. However, the relevance of the presence of APA to IVF outcome is controversial.

Indication for Testing

All patients experiencing two or more consecutive pregnancy losses, endometriosis, IVF failure suspected to represent implantation failure, premature ovarian failure, unexplained infertility, as well as patients who have systemic lupus erythematosus, or another collagen vascular disease should be tested for the presence of antiphospholipid antibodies (APA).

Detection Method

Phospholipid Antibodies to seven phospholipid antigens are detected using a solid-state ELISA Assay. The panel consists of 21 antiphospholipid antibodies that include IgG, IgM and IgA antibodies to cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid.

Specimen Requirements:

Collect one (1) 8 ml red or tiger top tube. Ship at room temperature overnight, next day morning.


 

Antithyroid Antibodies (ATA)

Introduction

Autoimmunity has been directly associated in the etiology of several reproductive conditions including unexplained infertility and recurrent pregnancy loss. Autoimmune thyroid disorders are characterized by the presence of antithyroid antibodies, specifically antithyroglobulin and antithyroid peroxidase (microsomal antibodies). Thyroid autoantibodies have been shown to be independent markers for pregnancies at risk for loss. Women who have antithyroid antibodies miscarry at approximately twice the rate of women who have no antithyroid antibodies. Individual levels of thyroglobulin and thyroid microsomal antibodies are similarly related to the increased miscarriage rate with no evidence of autoantibody specificity. Approximately 30% of women experiencing recurrent pregnancy loss have one or both antithyroid antibodies. Antithyroid antibodies have also been associated with implantation failure after in vitro fertilization (IVF) and embryo transfer (ET). Endometrial biopsies from women undergoing IVF with antithyroid antibodies show an increase in endometrial activated T cells and cells that secrete more Th1 cytokines and less Th2 cytokines than from women without antithyroid antibodies. An improvement in pregnancy rates after IVF/ET was seen in women with antithyroid antibodies after treatment with intravenous immunoglobulin.

Indication for Testing

All patients that have a history of thyroid abnormalities and/or a history of two or more pregnancy losses or unexplained infertility should be tested for the presence of antithyroid antibodies. Because women having antithyroid antibodies during the first trimester of pregnancy have a 50% chance of either miscarrying or developing thyroid dysfunction in her postpartum period, testing all women during the first trimester has been recommended.

Detection Method

Anti-thyroid antibodies are detected by standard ELISA assay.


 

Anti-Ovarian Antibodies (AOA)

Introduction

Anti-Ovarian Antibodies (AOA) have been reported in women with premature ovarian failure and unexplained infertility. Among women with infertility, those with evidence of ovarian autoimmunity appear to have poorer in vitro fertilization (IVF) treatment outcomes than women without antiovarian antibodies. Both reduced responsiveness to gonadotropin stimulation and reduced pregnancy rates are observed in women with ovarian antibodies. The frequency of antiovarian antibodies among women with premature ovarian failure varies between 35% and 69% depending on the patient population studied.

Indication for Testing

All women experiencing premature ovarian failure, having an elevated day 3 FSH level, experiencing unexplained infertility or experiencing a poor response to gonadotropin stimulation should be tested for the presence of anti-ovarian antibodies.

Detection Method

Anti-ovarian antibodies are detected by solid phase ELISA procedure


 

Anti-sperm antibodies (ASA)

Introduction

Male factor in infertility contributes to 40-60% of all cases. The term “immune male infertility” is used when “natural” formed anti-sperm antibodies bind to sperm surface antigens. Such antibodies can be detected in the male’s seminal plasma and/or serum and can be of IgG or IgA type. So far, there are 7 different antigen targets known on the sperm cell membrane: YWK-II, BE-20, rSMP-B, BS-63, BS-17, HED-2, and 75 kDa. In the presence of anti-sperm antibodies, the sperm cervical penetration is blocked or inhibited.

Indications for testing

Women and men with unexplained infertility, chlamidiasis or other uro-genital infection, men with sperm agglutinates and surgical interventions in reproductive organs should be tested for anti-sperm antibodies.

Detection Method

ASA in serum and seminal plasma are tested by ELISA method


 

Platelet-Leucocytes Aggregates (PLA)

Introduction

Formation of platelet-leukocyte-aggregates (PLA) represents an important mechanism by which leukocytes contribute to thrombotic events and platelets to inflammatory reactions. Damage to a blood vessel results in the adhesion of platelets to the site of injury and the subsequent formation of a platelet thrombus. Interaction of leukocytes with activated platelets is coordinated by an adhesion cascade in which P-selectin binds to P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. 

Purpose and methodology

Increased platelet-leucocyte aggregate has been reported in women that had RSAs or failed IVFs because of the thrombotic events that were diagnosed afterwards. This test can help not only for diagnostic purposes, but also to guide the physicians in order to perform the appropriate therapy with low molecular heparin for successful women’s pregnancy. This test can be performed in combination with the genetic thrombophilia panel testing. The methodology used for this test is flow cytometry and a green top tube is needed to run the test.