LABORATORY APPROACH TO SUBFERTILITY AND MISCARRIAGES Stella Prapa MD, PhD Microbiologist , Genetist

Subfertility and miscarriage are considered by many as a personal and social failure, failure of the woman and of the couple, and failure of the doctor and their mutual goal. In a time when medical science continually advances, in our center, we have found harmony between technological and scientific evolution on one hand and love and caring for our patient, on the other, for umanity medicine. For this reason, our center, following the evolution of medical science has enriched the laboratory- diagnostics department with a great number of examinations, ranging from basic microbiological tests to molecular genetics , with the goal of eugenics.

GENETIC REPRODUCTIVE PROFILE IN THE MAN Y Chromosome microdeletions DNA studies (AZF factor)‏ Cystic Fibrosis carrier screening (34 mut.) and Polymorphism 5T Kariotype (Chromosome analysis)‏ DNA fragmentation

SCREENING FOR RECURRENT SPONTANEOUS ABORTIONS OR RECURRENT MISCARRIAGES AND UNSUCCESSFUL IN VITRO FERTILIZATION TREATMENT Hormonal profile ( + thyroid)‏ Huhner test (antispermic antib.)‏ Kariotype‏

AMH ( Anti - Mullerian hormone )‏ Day 3 serum AMH levels reflect the size of early antral follicle cohort more accurately than other exams, as Inhibin B. The value of AMH measurements is great in the evaluation of ovarian responsiveness to exogenous gonadotrophins, follicular quality and also embryo implantation outcome. It can be used as a marker of ovarian being and as a marker for ovarian response in women undergoing IVF treatment and so it must be demanded after failure of the response of growing follicles to controlled ovarian stimulation.

Infections CMV Toxoplasma Rubella Listeria Herpes II Virus Parvovirus B19 Chlamydia Mycoplasma Ureaplasma

Thrombofilia PT, PTT Fibrinogen Antithrombin III deficiency Protein C Protein S APC resistance LA, ACL, ANA Preventive genetic testing molecular analysis (with PCR) - Factor V Leiden - Factor II, Prothrombin, mutation G20210A - Omocystein, mutation C677T of MTHFR gene

Immunological causes Autoimmunity Alloimmunity ANA APCA or cross match marker Anti-DNA AMA ASMA ASA (antispermic)‏ ATA (antithyroid)‏ RF (quantitative)‏ IgG, IgM, IgA APA (antiphospholipid syndrome) : ACL aPS aPE LA (Lupus anticoagulant)‏ NK, natural killer cells (CD3- , CD16+ , CD56+ )‏

TREATMENT FOR ALLOIMMUNITY Reflecting the controversies about causes and immune mechanisms of recurrent miscarriages, different and in part conflicting results have been published from different centres. Several authors have reported successful treatment by immunization of the prospective mother with paternal leukocytes. 75% (132/177)‏ 87% (132/152)‏ 25 20 132 Overall (n=177)‏ 89% (31/35)‏ 89% (31/35)‏ - 4 31 In early pregnancy (n = 35)‏ 73% (36/49)‏ 86% (36/42)‏ (7)‏ (6)‏ (36)‏ In ≥3 miscarriages (n = 49)‏ 71% (101/142)‏ 86% (101/117)‏ 16 101 Preconcep- tional (n = 142)‏ Success rate (A/A+B+C)‏ Successful pregnancy after Alloimmunity (A/A+B)‏ No pregnancy after ( C )‏ Alloimmunity‏ Abortions (B)‏ Births (A)‏ Alloimmunity time point Results of active immunotherapy in repeated miscarriages

26% (12/47)‏ 75% (12/16)‏ 31 4 12 Success rate (A/A+B+C)‏ Successful pregnancy (A/A+B)‏ No pregnancy after Alloimmunity ( C )‏ Abortions (B)‏ Births (A)‏ Results of active immunotherapy after unsuccessful IVF treatment Aside from the controversy about the immune mechanisms and effectiveness of alloimmunity and a possible psychological interaction with patients, immunotherapy with paternal lympocytes has 87% successful pregnancies in the examed overal group, and 86% in the patients with ≥ 3 abortions. So the positive therapeutic effect of alloimmunity in recurrent miscarriages is indisputable. End immunotherapy with husband's cells may modify the maternal immune response therefore immunization has been used to prevent further miscarriages.

A positive APCA test after alloimmunity in recurrent miscarriages predicts a successful pregnancy with a probability of 89%, making this test suitable as a predictive test Compared to passive immunotherapy with i.v. immunoglobulins , our therapy protocol with its immunization dose and frequency and its few side effects, proved to be better tolerated by the patients and more cost effective. As long as no therapeutic alternatives are available, patients with recurrent miscarriages should be offered alloimmunity with donor lymphocytes of the partner.