RECURRENT PREGNANCY LOSS (RPL) Definitions RPL Definition: Two to three consecutive spontaneous abortions (miscarriages) before 20 weeks gestation. Sporadic Abortion: A single pregnancy loss is a common event occurring in 10-20% of all human pregnancies. Incidence How Many Women Are Affected: Approximately 1-5% pregnant women have a diagnosis of RPL (40,000 - 200,000 U.S. couples/year). What Might Happen After Miscarriage: 49% of women with two consecutive losses and no live-born children will have a loss in their next pregnancy, whereas 29% of women with two losses and at least one live-born child will have a loss in their next pregnancy. Is Age a Factor in RPL: The incidence of a single miscarriage increases with female age in both normal and in RPL patients. The basic normal miscarriage rate is 10% per pregnancy ages 15-29 and as high as 55% for women >44 years old. This normal age dependent increasing miscarriage rate compounds the problem for women with RPL. POTENTIAL CAUSES OF RPL & DIAGNOSTIC TESTS The expert team of doctors, embryologists and nurses at RBA are highly adept at diagnosing, counseling, and treating simple and complex RPL conditions

1. PARENTAL GENETIC ABNORMALITIES The incidence of chromosomal abnormality in couples experiencing RPL is 3-5%. Diagnosis is made by chromosome testing (karyotype) of a patient’s blood sample. Most RPL patients with a chromosome abnormality have no physical evidence of a problem other than their history of RPL itself. Couples with a family history of genetic abnormality should be offered genetic counseling.

Figure 1 - Normal Male Karyotype (46 XY) from husband’s blood sample

Known and suspected types of parental chromosomal abnormalities causing recurrent pregnancy loss include: Translocation - A translocation occurs when genetic material is exchanged between chromosomes. The translocation is considered balanced if there is no net loss of genetic material and unbalanced if some material is lost during the translocation process. A significant percentage of genetically unbalanced (abnormal) sperm or eggs will form in translocation carriers. Thus, many adult translocation carriers will experience RPL and some will deliver babies with an unbalanced translocation causing mental retardation or other defects. Chromosome inversion - A less common chromosomal abnormality causing RPL is a chromosome inversion. This involves reinsertion of a segment of a chromosome in the reverse order following chromosome breakage. This can lead to duplications or deficiencies of genetic material during sperm or egg formation. Similar to translocations, carriers of inversions may produce unbalanced embryos causing RPL and other defects. Single-gene mutations - Mutations in genes required for embryonic, placental, or cardiac development may result in RPL. There are no specific tests for these genes now, but completion of the human genome project has set the stage for this area of molecular diagnosis.

Figure 2 - Female karyotype with abnormal exchange of genetic material between chromosomes 9:22 (Translocation)

Treatment Options for parental genetic abnormalities include the following: Pre-implantation genetic diagnosis (PGD) in combination with in vitro fertilization may allow some patients with genetic problems to conceive their own biological child. Since 1996, RBA scientists and doctors have achieved successful live-born pregnancies for many patients with chromosomal abnormalities. Donor sperm or donor egg may be appropriate for some couples with a genetic cause for RPL. 2. UTERINE ANATOMIC ABNORMALITIES It is estimated that 10-20% of RPL results from anatomic abnormalities of the uterine cavity. Abnormalities are diagnosed with an X-ray procedure (hysterosalpingogram - HSG) or an ultrasound procedure (sonohysterogram – SHG). Examples include: Congenital abnormalities Congenital abnormalities probably contribute to RPL through implantation failure or may cause late losses in some cases. Although some women can have normal outcomes with an abnormality in place, it is generally believed that loss rates approach 70% without correction. Septate Uterus - The most common uterine abnormality causing reproductive loss is the uterine septum. The septate uterus is corrected by hysteroscopic septoplasty, an outpatient surgical procedure. Patients with a uterine septum have excellent outcomes after repair with delivery rates approaching those found in the normal obstetrical population.

Figure 3 - HSG film of septate uterus

Figure 4 - Hysteroscopic Uterine Evaluation

Figure 5 - Uterine Septum Prior to Resection

Less Common Abnormalities: Cervical incompetence- Responsible for second trimester fetal loss, this diagnosis is based on clinical history of very early rupture of membranes without prior uterine contractions. Cervical cerclage is the treatment of choice. Occasionally, a surrogate is required. DES exposure- DES (diethylstilbestrol, a synthetic estrogenic compound) was incorrectly assumed to prevent miscarriage from the early 1950’s until 1973, when it was banned in the U.S. Exposure to diethylstilbestrol in uteri may be associated with RPL in “DES daughters”. DES exposure may result in T-shaped, unusually small (hypoplastic), or irregularly shaped uteri, cervical incompetence, ectopic pregnancy, and uterine constriction (hypoplasia). No good treatment other than surrogacy or adoption exists for patients with multiple confirmed losses without any other identifying cause. Acquired Uterine Abnormalities Myomas (Fibroids)- Fibroids are benign growths that can cause many reproductive problems, including pregnancy loss. Nonetheless, it is important to note that not all women with fibroids will experience reproductive problems. Four basic types of fibroids exist: pedunculated (on a stalk), subserosal (under the uterine surface), intramural (in the uterine wall), and submucosal (inside the uterine cavity). Myomas that distort the uterine cavity (usually submucus or large intramural types) may cause implantation failure, resulting from decreased vascularization of the endometrium, and should be surgically removed in RPL patients. Removal is accomplished via surgical myomectomy either via inpatient laparotomy (major surgery) or via outpatient hysteroscopic myomectomy (minor procedure).

Asherman’s syndrome- Scarring inside the uterus resulting from infection, retained products of conception, D&C, or previous uterine surgery. Treatment consists of hysteroscopic surgical correction. Several surgeries may be necessary to achieve a good result. Outcomes are dependent on the amount of original scar tissue present. At times, a gestational carrier may be necessary. A Note About Gestational Carriers (Third Party Reproduction) (Surrogacy) – For some patients presenting with a uterine problem, it is not possible to achieve a successful outcome through surgical means. Use of a gestational carrier may be recommended in these cases. Both in-state and out-of-state arrangements are handled on a routine basis at RBA. 3. HORMONAL & METABOLIC PROBLEMS: The incidence of endocrine abnormality in women with RPL is approximately 10-15% and may be much higher if patients with PCOS are included. Endocrine factors may include: Hyperprolactinemia – This disorder of pituitary prolactin oversecretion is usually caused by a small pituitary tumor. Diagnosis relies on hormonal measurements and exclusion of other causes of hyperprolactinemia (hypothyroidism, medication use). MRI imaging of the pituitary will reveal a small benign tumor in the majority of patients. Elevated prolactin may cause breast milk secretion (70% of patients), anovulation, and possible luteal phase defect. Treatment is directed by the diagnostic work-up of the disorder. Many patients will undergo therapy with one of several dopamine agonist medications. Thyroid dysfunction- Hypothyroidism is a common disorder found among reproductive age women. Diagnosis relies on demonstrating an elevated thyroid stimulating hormone (TSH) level. It is now believed that very minimal thyroid disease (sub-clinical hypothyroidism) may cause RPL and pregnancy complications nearly as much as more obvious thyroid disease. Treatment consists of normalizing thyroid function with thyroid hormone replacement therapy. Ovulatory problems and the possibility of luteal phase defect may be corrected by thyroid hormone replacement. Pregnancy loss rates are reduced to near normal levels when the disorder is corrected. Resolution may take several months after starting therapy. Calcium replacement with 500-1000mg supplementation while on thyroid replacement therapy is advised to lower the risk of osteoporosis during prolonged therapy. Diabetes mellitus – Untreated and undiagnosed, diabetes can cause severe birth defects and RPL. Diabetes is an infrequent cause of RPL. Normalization of blood sugar control prior to conception is mandatory. 4. ADVANCED REPRODUCTIVE AGE Defined as female age of 35 and above. Many women assume that reproductive success will naturally follow a healthy lifestyle. Unfortunately, living a healthy life will never change the basic fact that a woman is born with all the eggs she will ever have. The useful lifespan of human eggs is slightly more than half the normal human lifespan. Aging eggs have been subjected to a lifetime of unseen environmental reproductive toxins. A good number of women with unexplained infertility or RPL are experiencing accelerated reproductive aging. Aging eggs are more likely to be genetically abnormal. Thus, advancing female age (and increasing male age in some cases) will cause an increased number of genetically abnormal embryos. Diagnosis is based either on age alone on blood testing for a “day 3 FSH level” or the “clomid challenge test”, also known as the “CCCT”. These tests may uncover cases of abnormal ovarian function or “decreased ovarian reserve” occurring in women younger than expected. Although some patients with abnormal FSH test results will conceive on their own, many others require infertility therapy. Elevated day 3 FSH levels and reproductive aging – Both situations increase the risk of embryonic aneuploidy, adding to the risk of RPL and lowered pregnancy rates. Our physicians and nurses have a great deal of experience gained through counseling and treating the many patients with elevated FSH levels who continue to actively seek out advice and treatment at RBA. Treatment - Patients with “elevated Day 3 FSH” levels can be treated in one of three ways: Ovulation Induction – A woman will normally ovulate one egg per month during a natural cycle. If this single egg is abnormal, pregnancy will not occur or will result in a miscarriage. Use of ovulation induction drugs (clomid or gonadotropins) will generally increase the monthly egg yield so that a woman may have a better chance of having multiple eggs available for fertilization. Intrauterine insemination (IUI) is usually performed to further increase pregnancy rates. The problem with this therapy, especially in women >35, is that it relies on random chance to get a “normal egg” and only assumes (but does not prove) that at least one egg may be normal. Also, this therapy has not been proven to reduce miscarriage at all. Problems such as fetal Down’s syndrome cannot be screened prior to pregnancy as could be done with IVF/PGD. IVF using the patient’s own eggs – This technique is most useful if the resulting embryos can be tested with PGD. If PGD can be performed, this modality eliminates the majority of reproductive inefficiencies and allows us to select the best embryos for placement into the correct location. We are then able to get beyond some of the random chance issues of other therapies so that outcomes are enhanced. Donor egg IVF - RBA established the first egg donation program in Georgia in 1992 to serve the needs of patients with high FSH levels, impending reproductive failure, or actual menopause. Generally, success rates are much greater than seen with therapies using a patient’s own eggs. 5. POLYCYSTIC OVARIAN SYNDROME (PCOS) This disorder of androgen excess, anovulation, and altered insulin metabolism, “insulin resistance” is associated with up to a 50% miscarriage rate in patients <35 years old. Older patients may have a higher rate when the effects of PCOS and age are combined. Typical diagnostic tests include testosterone and DHEAS levels, fasting insulin/glucose ratio, FSH and LH levels, and ovarian ultrasound evaluation. Abnormalities, such as thyroid disease or hyperprolactinemia, should also be excluded. Patients with significant insulin resistance and high testosterone levels may be at greatest risk of miscarriage, as are obese patients. Treatment consists primarily of inducing ovulation to generate a pregnancy combined with use of Glucophage (Metformin) 1500mg daily. Use of Metformin has been shown to reduce the high miscarriage rate seen with PCOS to a level found in the general normal population (10-15%), if <35 years old. 6. IMMUNOLOGIC CAUSES OF RPL It is estimated that less than 5% of RPL is related to immune causes. There are two basic types of immunologic problems associated with RPL: Autoimmune (against self): This refers to the abnormal immunologic response of the woman to her own tissues. This response may subsequently carry over to produce RPL. Immune causes of this type include: Antiphospholipid syndrome (APS): Characterized by significant levels of antiphospholipid antibodies (APA, ACA) and /or lupus anticoagulant (LAC) and one or more clinical features, including RPL, intrauterine fetal death or thrombosis. APS is both an autoimmune and thrombophilic disorder. In general, antibody levels need to be of the IgG (rarely IgM) subtypes and found in moderate to high levels to be associated with RPL. APS is the cause of RPL in five percent of women and is most commonly associated with second trimester pregnancy loss. It may also cause very early loss in the form of implantation failure. Five percent of the normal population of reproductive women may be positive for low levels of antibodies associated with APS. Treatment of + ACA and/or +LAC patients consist of low- dose aspirin (75-81mg daily), beginning after ovulation and continued throughout pregnancy, combined with injectible Heparin 5000 units twice a day, usually beginning in the mid-luteal phase or with a positive pregnancy test. Alternative to Heparin includes Lovenox 40mg injections once daily. Alloimmune Response – an abnormal maternal immune response to pregnancy tissue (placental or fetal). A pregnancy has tissue unrelated to the mother that should induce a severe maternal immune response. Complex mechanisms exist to naturally prevent pregnancy rejection and problems in this system may occur. However, both investigation and treatment of these disorders is currently considered to be experimental. 7. LUTEAL PHASE DEFECT (LPD) LPD is a controversial cause of RPL. This can be a disorder of either low progesterone production by the ovary or low uterine response to progesterone. Historically, LPD was diagnosed following out–of–phase endometrial biopsies in at least two different cycles or by determining low luteal phase progesterone levels. However, both the endometrial biopsy and progesterone level testing have been shown to provide highly inconsistent results in numerous studies. Therefore, physicians oftentimes differ in their diagnostic evaluation of this disorder. Alternatively, it is entirely appropriate to not attempt diagnosis at all and proceed with treatments that may potentially correct a possible LPD. Treatment primarily consists of inducing ovulation to produce improved egg production (folliculogenesis) which, in turn, will correct a luteal phase defect in the majority of patients. Patients may also be administered progesterone supplementation in addition to ovulation induction. 8. TOXIN EXPOSURE Toxins such as ionizing radiation, mercury, lead and alcohol are confirmed teratogens, and could contribute to pregnancy loss. Hyperthermia (fever or high body temperature during early pregnancy) is a suspected teratogen. Cigarette smoking has been linked to miscarriage, ovarian damage, diminished ovarian reserve and possible earlier than expected menopause. The teratogenic effect of pesticides is unknown. It is doubtful that exposure to food additives, artificial sweeteners, hair dye, nail polish, and herbicides contribute to recurrent pregnancy loss. It is advisable to eliminate toxins at least three months prior to conception. 9. RECURRING EMBRYONIC ANEUPLOIDY Approximately 50% of couples experiencing RPL have no cause identified after routine testing. Many of these patients may be experiencing recurring embryonic genetic abnormalities incompatible with normal survival (aneuploidy). Examples of this situation frequently treated by RBA physicians include patients who are referred to us for continued losses despite appropriate treatment of other non-genetic causes of RPL. A significant number of these patients were subsequently found to have nearly twice the expected rate of embryonic genetic abnormality during IVF/PGD. The risk of chromosome abnormalities (aneuploidy) in embryos increases primarily with female age, and to a much lower extent, with increasing male age. However, with the increasing use of IVF/PGD, we have seen very high rates of embryonic aneuploidy in previously unexplained RPL patients starting as young as their mid to late 20’s. Thus, recurring embryonic aneuploidy appears to be a common cause of RPL in patients where no other cause has been identified regardless of age. Diagnosis is accomplished by either testing placental tissue obtained from an actual pregnancy loss specimen or by generating early embryos through in vitro fertilization (IVF) and testing one to two cells from each embryo with preimplantation genetic diagnosis (PGD). A few select embryos with normal results are then transferred into the patient’s uterus. PGD - Since 1996, RBA has been a national leader in the field of IVF/PGD applied to many causes, including RPL. A 2004 RBA study with forty RPL patients undergoing PGD uncovered a very high 69.3% embryonic abnormality rate. Nonetheless, a 50% pregnancy rate was obtained for these high-risk patients (avg age = 35.4 years old) when two genetically tested embryos were replaced. 10. THROMBOPHILIC DISORDERS (Excessive blood clotting) Hypercoagulable conditions that are either inherited or acquired may cause RPL and pregnancy complications. Testing is generally performed if no other cause of RPL can be identified or if there is a significant family or personal history of thrombosis or early cerebrovascular events (stroke). Prior unexplained fetal death (loss of heartbeat after 8 weeks), severe fetal growth restriction with no other identifiable cause, and early toxemia (preeclamsia) during pregnancy would be another indication for testing. Diagnosis is based on blood testing for Factor V Leiden mutation, Prothrombin gene mutation, Protein C and S deficiency, and hyperhomocysteinemia (MTHFR mutation). Factor V Leiden, Prothrombin Factor II, Protein C&S deficiency, Antithrombin III deficiency, LAC/ACA - We treat these disorders with low- dose aspirin and Heparin 5000 units subcutaneously twice a day (or Lovenox injections 40 mg sq/day). Live-born rates are approximately 70- 80% overall. Hyperhomocysteinemia (MTHFR mutation) – Treated with increased folic acid (2-5 mg daily, vitamin B6, B12 – Folgard Rx) prior to and during gestation. 11. MALE REPRODUCTIVE ABNORMALITIES Males with significant, persistent abnormal sperm morphology (abnormal shape) may father a higher number of genetically abnormal embryos. Diagnosis requires several semen analyses. We also test sperm with the DNA fragmentation analysis which may also predict the presence of a higher than average percent of genetically abnormal sperm. It is possible that some cases of RPL may be related to increasing male age – particularly in men over fifty years old. Treatment - RBA is one of the few fertility centers in the U.S. to have a full-time, board-certified male factor reproductive specialist on staff. Varicocele repair has been shown to improve sperm morphology but it is uncertain if RPL rates can be diminished as a result of this corrective procedure. Use of antioxidants and vitamins may also improve sperm parameters but have not been proven to generate an improved clinical outcome. A new sperm selection device, the PICSI dish, has been evaluated at RBA for use during IVF/ICSI. It remains unclear if it will allow selection of more genetically normal sperm. It also remains unclear if use of intrauterine insemination (IUI) will select more normal sperm and subsequently improve pregnancy outcomes in RPL patients with associated male factor. 12. INFECTIOUS CAUSES OF RPL Although maternal infections, such as cytomegalovirus (CMV), toxoplasma, herpes and rubella have been implicated as causes for single pregnancy losses, they are not implicated in RPL. The same conclusions apply to mycoplasma, ureaplasma, syphilis, and listeria infections as causes of human RPL. Therefore, many physicians may elect to exclude these tests in the RPL evaluation. Bacterial infection within the uterine cavity can be diagnosed with endometrial biopsy and treated with antibiotics. Oftentimes, a two-week course of antibiotics are prescribed to RPL patients when no other cause can be found. OUTCOMES WITHOUT MEDICAL THERAPY Approximately 50% of patients experiencing RPL will have a cause identified through routine medical testing. A higher percentage of abnormalities will be detected if IVF/PGD is performed. Approximately 70% of couples experiencing RPL will have a liveborn child without medical therapy. Most of these patients will be < 35 years old. Benefits of medical therapy: Provides assistance to the 30% of couples destined to have a low chance of a liveborn child without therapy. Helps to shorten the time interval to a successful outcome. May lower the risk of more losses before a successful outcome. Psychological Aspects and Support Measures The physicians and staff at RBA are sensitive to the very unique needs of our RPL patients. We are here to advise you and to listen to your concerns. Fully trained psychological counselors are available for you to consult with as part of your treatment protocol.