The investigation of each couple facing infertility problems requires a meticulous access which should be performed and evaluated by specialists in the field of Assisted Reproduction.

After obtaining a complete previous medical history from both partners (which should comprise among others questions on previous diseases, surgical operations and pregnancies, on the duration and features of the woman’s menstrual cycle, on the frequency of sexual intercourse and the time period of conceiving efforts) and performing a clinical gynecologic examination, the fundamental questions that should be answered in any case before the application of any simple or complicated assisted reproduction technique on a couple are the following: is the man’s sperm capable to fertilize? do the woman’s ovaries release an oocyte (egg) every month? are the woman’s tubes open enough to allow the spermatozoa and the oocyte to meet? Beyond this stage, any further investigation should be specialized according to each specific situation.

• Diagnostic investigation of the male: The main test for the male investigation is the assessment of sperm (semen analysis) which is recommended to be performed after a 3-day abstention from sex, while its collection by means of masturbation should preferably be done in the laboratory (if the collection is done at home, the sample must be kept warm and brought to the laboratory within 30 minutes).

In cases that no definite aberrations are observed, it is usually recommended that the examination should be repeated after a short period of time. The semen analysis gives information on its volume and acidity, on the number, morphology and motility of spermatozoa, on the presence of inflammatory cells, and on its glucose and other substances content. On suspicion of infection a semen culture follows, while in the suspicion of antisperm antibodies a series of specific detection examinations may follow. Finally, it is possible to determine by means of specialized tests the functional ability of spermatozoa to penetrate the oocyte in order to fertilize it. On suspicion of varicocele, an ultrasonographic scan of the scrotum is performed to detect dilated veins. On suspicion of endocrine disorder, hormone tests are performed (serum testosterone, FSH, LH evaluations, etc). Finally, in cases of severe oligospermia or azoospermia a chromosomal analysis (caryotype) as well as a genetic test for cystic fibrosis should be done.

• Diagnostic investigation of the female

The investigation for female factor infertility comprises a series of essential and optional tests.

The hysterosalpingogram (HSG) is a test that should be done to women before their entrance to any assisted reproduction programs as this provides valuable information on the status of the uterine cavity and tubes. The HSG –which should be done 3 to 6 days after the end of menstruation- includes the injection of a contrast dye through the cervix, while continuous x-ray pictures are taken at subsequent stages in order to detect whether the dye is prevented or not from passing through the tubes towards the pelvis. In this way we can detect any abnormalities found into the uterine cavity such as septa, sub-mucous fibroids or adhesions as well as tubal abnormalities such as lumen dilation or obstruction of the fallopian tube. It must be noted that even if it is decided for a couple to proceed to in vitro fertilization straightforward, a HSG should be performed as the presence of a hydrosalpinx for instance, may have a negative impact on the IVF results and thus its removal should be suggested before any IVF attempt commences.

The ultrasound scan is an indispensable diagnostic examination. It is a safe imaging method using ultra-frequency sounds (ultrasounds) which are transformed into images by means of advanced technology. An ultrasound scan can be performed either through the abdominal walls (transabdominal ultrasonography) or through the vagina (transvaginal ultrasonography); the most advanced ultrasonography devices offer impressive high-definition images. This method is used to visualize the female pelvic organs to detect any possible deviations from normal anatomy of the area, like ovarian cysts, hydrosalpinx, uterine fibroids, endometrial polyps, etc.

In order to detect ovulation, numerous direct and indirect ways are available. Firstly, the woman’s medical history alone may demonstrate that there is no ovulation problem provided that all menstrual cycles are normal and regular (lasting from 25 to 34 days). A simple, traditional method of detecting ovulation is the body temperature measurement using a common thermometer every morning right before the woman gets out of bed. One day after ovulation there is a slight temperature rise which persists until the beginning of the next menstruation. This method is simple but has the disadvantage that body temperature may be affected by many other factors leading to false conclusions. Progesterone measurement during the 21^st day usually gives increased values of this hormone provided that this is preceded by an ovulation. Moreover, consecutive measurements of another hormone (luteinizing hormone, LH) in the woman’s blood or urine reveal increased values a few hours before ovulation. Finally, consecutive ultrasonograms may be used to monitor the predominant follicle’s growth during the menstrual cycle and to confirm its rupture if and when ovulation occurs. The ultrasonogram also helps in detecting any other possible disorders in the female reproductive system such as uterine fibroids, ovarian cysts, etc.

Endocrinological investigation is performed in women on clinical suspicion of endocrine disorders like hirsuitism, polycystic ovarian syndrome, menstrual cycle disorders, or in the case that we intend to roughly evaluate the ovarian reserve in oocytes in order to draw the scheme and intensity of stimulation. In order to provide reliable information, the hormone evaluations must be performed at specific cycle days and include on occasion measurements of blood estrogen, progesterone, androgens, pituitary releasing hormones (FSH, LH), prolactin, thyroid hormone levels, etc.

In cases of doubt or detection of pathology after performing a hysterosalpingogram or ultrasonogram, there may be need to proceed to the performance of hysteroscopy and/or laparoscopy as parts of the diagnostic investigation. A hysteroscopy, which is performed during the first days after the end of menstruation, gives us a precise visualization of the cervical canal and the uterine cavity together with the tubal ostia. The hysteroscopy (as this is performed in the ‘Embryo ART’ Unit) is a simple and almost painless procedure for the woman. The mildest form of sedation is administered (the woman is able to watch the operation through a monitor); then the hysteroscope (a long, thin pipe) is inserted through the cervix with no use of any other instruments and without performing any cervical dilatation (which should be better avoided in any woman and especially in those trying to get pregnant), until it advances to the uterine cavity which is simultaneously filled with normal saline to enable real-time monitoring. In case of pathological findings (endometrial or endocervical adhesions, endometrial or endocervical polyps, uterine septa), those may be fixed at the same time with the use of advanced micro-instruments that are inserted through the hysteroscope. The woman may return to her everyday activities immediately after the process. Ideally, a hysteroscopy should be performed within the basic investigation before the application of assisted reproduction techniques; in practice it is usually performed (and this should be the case) after 2 or 3 unsuccessful embryotransfers or of course on suspicion of endometrial pathology after the ultrasonographic or hysterosalpingographic investigation. By performing laparoscopy the entire pelvis of the woman can be investigated in order to detect adhesions or endometriosis or to evaluate the status of fallopian tubes, or also to investigate whether the tubes are open or closed by injecting a blue dye through the cervix; in case that there are no obstructions the blue dye flows out of the tubes into the pelvis. A laparoscopy is always performed in an operation room under general anesthesia with the use of the appropriate instrumentation. The woman’s abdomen is filled with gas, and the laparoscope is introduced into the abdomen through a small cut made near the umbilicus. The picture is projected on a monitor through a camera, allowing an accurate visualization of all pelvic organs. Through 2 or 3 more cuts, additional instruments are inserted through the abdominal wall to allow the performance of gentle moves as well as the performance of an operation (removal of ovarian cysts, lysis of pelvic adhesions, salpingectomy, destruction of endometriosis spots, etc).

The examination of cervical mucous after sexual intercourse has been applied to investigate cervical factor infertility. One or two days before the expected ovulation, the couple has sexual intercourse and 2 to 12 hours later the woman comes to the unit for a cervical mucous sample to be taken. The mucous is then examined under a microscope to investigate the presence or not of specific expectable features as well as of motile spermatozoa. Thus we acquire data on the necessary cooperation between male and cervical factor for this specific couple.

Finally, both partners of every couple preparing to undergo any assisted reproduction technique must undergo tests for β-thalassemia trait, hepatitis B surface antigen and core antibody, hepatitis C antibody, and HIV.