The cryogenic laboratory is an integral part of KRIOBANK’s Infertility Treatment Centre, dedicated to the cryopreservation of biological materials. From the point of view of patients being treated for infertility, the most important cryogenic applications are cryopreservation of sperm and isolated reproductive cells (Sperm Bank), and embryos (Embryo Bank). The ability to use donor semen, depositing your own semen for later use, and freezing and storing embryos are the most common services we provide during infertility treatment.
Cryopreservation and embryo storage
Often our patients are men who, after several semen analyses and testicular biopsy are diagnosed with azoospermia. This situation completely excludes the possibility of pregnancy. Azoospermia, except for cases caused by obstruction of the seminal tract (obstructive azoospermia) or hypogonadotropic hypogonadism, is usually irreversible, and conservative treatment has no positive effect. In this situation it is possible to use anonymous donor banks. KRIOBANK, as the first and currently one of few institutions in Poland, offers help to couples in whom the man does not produce sperm. Donor sperm may be used for insemination or IVF treatment.
It has been proven that sperm can be stored at very low temperatures for many years, without losing the ability to fertilize. This is extremely important for men whose fertility may be threatened in the near or distant future. This would most likely occur due to treatment of testicular cancer or other organs and systems that require the use of chemotherapy or radiant energy. Very often, side effects of such treatment are significant impairment of the male reproductive function, or complete and irreversible infertility. There are also other clinical situations where treatment has a high risk of complications, resulting in significant impairment or damage to reproductive functions. A typical example is testicular atrophy occurring after undergoing a varicocelectomy.
When these situations occur in young men who have not yet completed their reproductive plans, it is possible to deposit some semen, preferably a few batches, for storage. It will await the right time to start a family, providing a chance for having one’s own offspring. It is preferable for the sperm to be deposited prior to starting primary treatment. In some men with critically low semen parameters, sperm appear sporadically, once in a while, which makes access to them “on demand” difficult. When planning in vitro treatment in this situation it is necessary to obtain sperm earlier and freeze them to ensure treatment will be possible. In this case we use unique methods of cryopreservation of single, live sperm in ovarian casings.
For long-term sperm storage it is necessary to stay in constant contact with KRIOBANK and inform us of changes in address, as well as confirm annually the request to store the deposit.
A routine KRIOBANK practice is the short-term deposition of semen by all patients being prepared for treatment. This ensures that it is available at all times, and provides the opportunity to complete treatment without the partner’s presence, as well as protect against the unexpected deterioration of its quality.
Cryopreservation and embryo storage
Approximately 30% of IVF patients receive more embryos than can be used in one transfer. This is due to the following facts. The number of embryos transferred to the uterus is limited to one or two at a time. This is to prevent multiple pregnancies in accordance with the recommendations of all world organisations and expert bodies. Unused embryos are reared for five days and once they exhibit normal late embryonic development (reaching the blastocyst stage), they are cryopreserved. Freezing early embryos (up to the second day) is not recommended because most of them stop developing due to developmental disorders, mainly (85%) of genetic origin. Cryopreservation of embryos is a very complicated procedure that requires the use of advanced methods and equipment.
Frozen and stored embryos are the exclusive property of patients (biological parents) and await their decision as to the transfer. Most often it is postponed, because in the group of patients who froze embryos the efficacy of treatment reaches 60%. During their storage period, parents must contact Kriobank regularly, informing them of any changes to their addresses and telephone numbers.
In vitro patients have the right to limit the number of fertilized oocytes, which eliminates the possibility of excess embryos, and hence the need to freeze them. We respect this decision and adhere to the requests of future parents.
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Znaczny odsetek naszych pacjentów, po wstępnej diagnostyce jest kwalifikowany do leczenia zachowawczego, które polega na obserwacji cyklu, indukcji jajeczkowania, ocenie jakości owulacji oraz wyznaczeniu najlepszego czasu dla rozrodu naturalnego. Należy podkreślić, że każde zastosowanie leków indukujących jajeczkowanie powinno być poprzedzone wizytą lekarską w czasie pierwszych dni krwawienia miesięcznego.
Ma ona na celu ocenę stanu wyjściowego jajników, co umożliwia:
wykrycie sytuacji stanowiących przeciwwskazanie do wdrożenia leczenia,
uniknięcie nieuzasadnionego użycia leków (ciąża) oraz ocenę ryzyka ich zastosowania.
Przy braku przeciwwskazań programujemy użycie leków, określamy termin prawdopodobnej owulacji oraz sposób monitorowania odpowiedzi w zakresie liczby, stopnia rozwoju oraz jakości pęcherzyków przedowulacyjnych. Umożliwia to maksymalne zwiększenie szans rozrodczych oraz uniknięcie powikłań w postaci ciąży mnogiej jak i zespołu hiperstymulacyjnego. W monitorowaniu przebiegu owulacji stosuje się przezpochwowe badanie USG oraz ocenę stężeń estradiolu oraz progesteronu, co wraz z wizytą lekarską zajmuje około 2 godzin.
W warunkach naturalnego rozrodu częstość występowania ciąż mnogich szacuje się na 1,9%, chociaż wydaje się, że ten wskaźnik może być zaniżony wskutek naturalnej embrioredukcji jednego płodu i donoszeniu drugiego. Użycie przezpochwowej USG o wysokiej rozdzielczości, we wczesnym okresie naturalnej ciąży pozwoliło określić częstość ciąż mnogich na 12%. Ciąże bliźniacze donoszone do terminu porodu stanowią zaledwie 2% wszystkich porodów. Samoistna embrioredukcja ciąży bliźniaczej zakończona porodem jednego płodu ma miejsce w 12% przypadków. Poród naturalnej ciąży trojaczej w krajach zachodnich odbywa się raz na 8000-9500 porodów. Odsetek poronień w grupie ciąż bliźniaczych sięga 20%, a w ciążach wielopłodowych jest znacznie wyższy. Powikłania położnicze (preeclampsia, krwotoki położnicze, choroba zakrzepowo-zatorowa), poród przedwczesny oraz związana z nim wysoka umieralność noworodków, jak również wcześniactwo, nierozerwalnie kojarzące się z ciążą mnogą, dopełniają całości problematyki i stanowią o żywym zainteresowaniu problemem środowisk ginekologów i położników.
W ostatnich 30 latach problem ciąż mnogich przybiera na znaczeniu w związku z postępem medycyny i rozwojem metod leczenia niepłodności. Jatrogenne ciąże mnogie są wciąż jednym z najczęstszych powikłań leczenia. Należy jednak stwierdzić, że dwie trzecie takich ciąż powstaje w wyniku leczenia zachowawczego, związanego z indukcją jajeczkowania dla potrzeb naturalnej prokreacji lub zabiegów inseminacji. Jedynie jedna trzecia jest wynikiem zaawansowanych technik wspomaganego rozrodu (ART), których klasycznym przykładem jest program zapłodnienia pozaustrojowego oraz transferu zarodków (IVF-ET).
Fakt zaistnienia ciąży bliźniaczej często spotyka się z akceptacją ze strony pary małżeńskiej, zazwyczaj zmęczonej i zestresowanej długim okresem oczekiwania na dziecko. Jednakże ciąża wielopłodowa zawsze stanowi niezwykle dramatyczną wiadomość i w obliczu danych na temat powikłań i zagrożeń, związanych zarówno z przebiegiem ciąży jak porodu oraz perspektyw zdrowotnych potomstwa zmusza do rozważenia embrioredukcji. Należy zauważyć, że polskie prawodawstwo nie dopuszcza interwencji medycznej tego rodzaju.
Podstawową rzeczą jest identyfikacja pacjentek, narażonych na wystąpienie ciąży wielopłodowej. Do czynników predysponujących zalicza się młody wiek pacjentek, a w badaniu ultrasonograficznym obraz policystycznych jajników. Kolejnymi czynnikami ryzyka, pojawiającymi się już podczas hormonalnej indukcji owulacji, są liczba wzrastających pęcherzyków oraz wartość maksymalnego stężenie estradiolu w surowicy krwi. Przy wartościach estradiolu powyżej 2000 pg/ml, jak również w przypadku wzrastania więcej niż 4 pęcherzyków przekraczających średnicą 17 mm, u pacjentek przygotowywanych do inseminacji wewnątrzmacicznych powinno się rozważyć wykonanie punkcji odbarczającej jajników, usuwając nadmierną liczbę pęcherzyków, bądź też wykonanie programu zapłodnienia pozaustrojowego. Wobec braku możliwości technicznych dla takiego postępowania należy odstąpić od próby uzyskania ciąży w danym cyklu i podjąć ponowną indukcję owulacji w kolejnym cyklu, tym razem z bardziej wyważoną dawką leków. W przypadku zapłodnienia pozaustrojowego praktyka transferowania tylko jednego zarodka wyeliminowałaby ryzyko ciąży mnogiej, ale także znacznie ograniczyłaby szanse na posiadanie dziecka. Wiadomo bowiem, że implantacyjność wczesnego ludzkiego zarodka sięga 14-20%. Tak wiec przy transferze dwóch zarodków, szansa na ciążę wynosi około 30-40%. Niski wskaźnik powodzenia podczas przeniesienia jednego zarodka byłby trudny do zaakceptowania zarówno dla pacjentów jak i lekarzy, a całościowy koszt leczenia wzrósłby niepomiernie. Według najnowszych rekomendacji Światowej Organizacji Zdrowia nie powinno się transferować więcej niż dwa zarodki w jednym cyklu leczniczym. Odpowiednio funkcjonujący program mrożenia zarodków oraz późna ich hodowla i transfer blastocyst, umożliwiają realizację tego postulatu bez zmniejszenia odsetka ciąż. W naszym ośrodku od wielu lat stosowaliśmy zasadę transferu dwóch zarodków. Jedynie w przypadku kobiet powyżej 35 roku życia, przy złej jakości zarodków oraz przy wielokrotnych niepowodzeniach leczenia, przenosiliśmy trzy embriony. Ta strategia pozwoliła nam ograniczyć liczbę ciąż mnogich (patrz „Wyniki uzyskiwane w Kriobanku”), jednak nie ustrzegliśmy się ciąż trojaczych. Wśród 5 takich przypadków, 3 zakończyły się niepowodzeniem. Przeżyliśmy razem z naszymi pacjentami te tragedie i podjęliśmy decyzję o całkowitym odstąpieniu od transferu 3 zarodków od połowy 2002 roku. Mamy dowody, że ta strategia ograniczyła jeszcze bardziej odsetek ciąż bliźniaczych i wyeliminowała całkowicie ciąże trojacze różnojajowe. Ten sposób postępowania rekomendują wszystkie organizacje zrzeszające ośrodki leczenia niepłodności, mając na uwadze fakt, że odsetek ciąż pojedynczych stanowi najlepszy parametr stanowiący o jakości leczenia (parameter of excellence).
Zespół hiperstymulacji jajników
Pojawia się w następstwie indukowania owulacji przy użyciu leków, w szczególności gonadotropin. W prawidłowym cyklu miesięcznym dochodzi z reguły do powiększenia jednego pęcherzyka w jednym jajniku, z którego uwalnia się podczas owulacji jedna komórka jajowa. W wyniku działania leków hormonalnych możliwa jest indukcja wielu pęcherzyków w obu jajnikach. Wystąpienie tego powikłania wiąże się z nadmierną odpowiedzią jajników na leki. Wobec nieznanej etiopatogenezy oraz braku skutecznego leczenia, szczególnego znaczenia nabiera zapobieganie jego wystąpieniu. Można to osiągnąć poprzez: precyzyjne rozpoznanie warunków wstępnych, indywidualizowanie dawki leków, uważne monitorowanie przebiegu leczenia, przerwanie leczenia, odstąpienie od transferu zarodków, zmniejszenie dawki HCG u pacjentek szczególnie zagrożonych. Najczęstszymi objawami są silne bóle brzucha, obrzęki, wzdęcie brzucha oraz czasem objawy duszności. Objawy te wynikają ze znacznego powiększenia jajników oraz gromadzenia się płynu w wolnych jamach ciała i mogą stopniowo nasilać się stanowiąc czasem zagrożenie życia. Mogą występować w trakcie lub bezpośrednio po stymulacji (wczesny zespół) lub też kilka dni po jej zakończeniu, jako konsekwencja rozwijającej się ciąży (późny zespół). Powikłanie to, w postaciach ciężkich jest groźne dla życia i wymaga intensywnej opieki w warunkach szpitalnych. W związku z tym ośrodek zajmujący się leczeniem niepłodności małżeńskiej za pomocą technik wspomaganego rozrodu powinien zapewnić ciągłość leczenia w razie wystąpienia powikłań.
Kontrolowana hiperstymulacja jajników dla potrzeb zapłodnienia pozaustrojowego w sposób szczególny wiąże się z ryzykiem wystąpienia zespołu hiperstymulacyjnego, w związku z czym wskazane jest stosowanie mniej agresywnych form leczenia. Celem takiego postępowania jest uzyskanie umiarkowanej liczby komórek jajowych i poddawanie zapłodnieniu jak najmniejszej liczby oocytów, która jednak zapewnia akceptowalną szansę na ciążę. W określeniu tej liczby powinno być pomocne, uwzględnienie wyników uzyskiwanych w podobnych grupach pacjentek, poddanych analogicznym protokołom i dawkom leków.
Classic indications for IVF treatment include blocked Fallopian tubes, moderate male factor, and ineffectiveness of previously used treatments. This method was developed by a team of British doctors and embryologists led by Edwards and Steptoe. As a result of their work, on 25 July 1978 in England Luise Brown was born – the first human child born as a result of IVF treatment and transfer of the developing embryo into the uterine cavity. The effectiveness of the first trials, conducted in the natural cycle, was relatively low. Only the introduction of ovulation stimulation and soon after controlled ovarian hyperstimulation, significantly increased it.
Thanks to this, IVF treatment has gained worldwide recognition. After nine years, on 12 November 1987, at the Institute of Obstetrics and Gynaecology in Białystok, the first child conceived as a result of in vitro fertilization performed in Poland was born. Currently, IVF is performed throughout the world, and indications for its use include almost all causes of infertility.
Indications for IVF treatment according to the latest recommendations of the Polish Society of Reproductive Medicine include:
1) Fallopian tube factor*
a) in patients with permanent damage to fallopian tubes
b) in patients not qualifying for surgery
c) in patients with impaired function of fallopian tubes with retained patency, or after microsurgical surgery and 2 years without pregnancy; It is recommended to wait when there are no other factors that may affect the chance of pregnancy (abnormal semen, woman’s age > 35 years, duration of infertility <3 years, endometriosis, ovulation disorders)
2) Unexplained infertility**
a) lasting longer than 3 years
b) if the patient is over 35 years of age – sooner
3) Male factor
a) total number of mobile sperm <1 million – ICSI recommended
b) number of mobile sperm is 1 – 10 million – in case of infertility lasting longer than 2 years**
c) sperm count > 10 million – as in unexplained infertility
a) degree I, II, as in unexplained infertility
b) degree III, IV, as in fallopian tube factor
5) Hormonal Disorders**
a) 12 stimulation cycles without effect
6) Unsuccessful attempts at intrauterine insemination
a) maximum of 6 trials <35 years of age
b) a maximum of 4 trials > 35 years of age
*It is advisable to remove the fallopian tube in case of hydrops
** 4-6 intrauterine inseminations should be considered before IVF/ICSI treatment
7) Currently, IVF treatment is also used in fertile couples, where:
a) the male partner is HIV or HCV positive and the female partner is not infected
b) the couple is a carrier of genetic modifications that cause severe, irreversible changes in the offspring, and pre-implantation diagnostics avoids a difficult decision to terminate a pregnancy
c) female partner begins cancer treatment and treatment is likely to irreversibly damage the ovaries
d) ovulation induction results in a large number of developing follicles and this situation poses a high risk of multiple pregnancy.
Course of treatment
Course of treatment
IVF treatment can be divided into several stages.
– Pharmacological ovarian stimulation
The first treatment consists of pharmacological ovarian stimulation for the growth and maturation of Graaf follicles. Most infertility researchers believe that this process is a key component of treatment. The end result of treatment largely depends on the success of ovulation induction. In the most commonly used stimulation regimens, GnRH analogues are administered simultaneously with gonadotropins. The stimulation process requires an ultrasound assessment (assessment of the number and diameter of growing follicles), and determination of serum estradiol (E2) concentrations as the index for the endocrine function of developing Graaf follicles. When the dominant pre-ovulation follicles reach the size of > 18 mm and the average E2 concentration of 150-200 pg/follicle, the intramuscular administration of 10,000 units of hCG – human choriogonadotropin simulates an exogenous luteinizing hormone (LH).
– Ovarian puncture
Egg collection takes place 34-36 hours after hCG administration, by puncture under ultrasonography. For this purpose, transvaginal probes (5-7.5 MHz) are used with a guide used for procedures performed via the posterior vaginal vault. The procedure involves searching for eggs in the resulting follicle fluid and transferring them to the culture medium. The ovary is surrounded by the oocyte-corona radiata-cumulus complex (OCCC). OCCC morphology, in assessing the degree of the dispersion of the cumulus oophorus and the corona radiata, is the basis for determining egg quality and maturity. Genetically immature ovaries, those in the germinal vesicle (GVBD) and germinal vesicle breakdown (GVBD) phases, are not capable of being fertilized.
– Egg fertilization and embryo development
Sperm used during in vitro fertilization are prepared using the ascending migration or the discontinuous Percoll gradient centrifugation method. Before insemination, the ova are incubated for about 3 hours, which allows them to complete the maturation process. Approximately 100,000 sperm are added to the oocytes placed in the culture plate, and after 19 hours of incubation, the number of pronuclei (PN) is evaluated. Their presence testifies to a successful fertilization. Correctly fertilized eggs will have two pronuclei, and in the perivitelline space there should also be two polar bodies present. Fertilized cells are transferred to a new surface. Approximately 5-10% of cells are fertilized incorrectly, as evidenced by the presence of more than two pronuclei. They should be eliminated from further cultivation. About 28-32 hours after fertilization the first division of the embryos occurs and two blastomeres are visible. On the second day the embryos consist of 3-5 cells, after 48-72 hours they reach the stage of morula-blastocyst. The embryos may be transferred to the uterine cavity at 2, 3 or 5 days after fertilization. In the morphological classification of embryos, the rate of cell division, the symmetry of blastomeres and the extent of their fragmentation are evaluated. The greatest development potential is shown by fast-dividing embryos, containing equal blastomeres, showing no fragmentation. The chance of getting pregnant decreases with increased morphological abnormalities in the embryo.
– Embryo transfer
In the past, anywhere from one to four, and sometimes even more embryos would be transferred to the uterine cavity, depending on the adopted norms of a given country and clinic. It has now become standard to transfer a single embryo to women up to the age of 35, and up to two embryos to older women. This standard is also recommended by the Polish Society of Reproductive Medicine, and followed by us at KRIOBANK. The trans-cervical transfer of embryos to the uterus is done by using special catheters. Embryos not used for transfer are cryopreserved at the early stage of embryonic development (4-6 blastomeres), or after the blastocyst stage in prolonged culture.
-Supplementation of the luteal phase
Experts agree that, as a result of the IVF treatment, luteal failure occurs as a consequence of defects in corpus luteum production. Among the reasons for this are the aspiration of granular cells during ovariectomy, as well as the low LH concentration resulting from GnRH agonists. Hence the accepted practice is supplementation with gestagens during the second phase of the cycle. During the second phase of the cycle progesterone is administered intramuscularly at a dose of 25 – 100 mg per day, or intravagally at a dose of 90-600 mg per day. Another type of supplementation is oral dydrogesterone (Duphaston) or estradiol valerate with 17β-hydroxyprogesterone caproate (Gravibinon) administered intramuscularly. Supplementation of the second phase of the cycle is also possible with hCG. In controlled studies, the highest efficacy was demonstrated by the intramuscular use of progesterone and hCG, as well as intravaginal progesterone supplementation.
Intracytoplasmic sperm injection - ICSI
Intracytoplasmic sperm injection – ICSI
The Intracytoplasmic sperm injection (ICSI), introduced more than 10 years ago, involves the direct microsurgical introduction of male gametes into the cytoplasm of the egg. This allows to bypass most of the stages of natural gamet interaction, and thus eliminate some of the causes of infertility associated with the male factor. The most common indications are low sperm count (oligospermia), impaired motility (asthenospermia), and abnormal teratozoospermia (OAT – oligoasthenoteratozoospermia). Also, ICSI is the only method in the absence of spermatozoa (azoospermia), both obstructive and non-obstructive, as well as ejaculatory disorders and necrospermia.
In terms of ovarian induction and obtaining female gametes, ICSI does not significantly differ from classical IVF. For men whose semen contains sperm, semen preparation involves classical preparatory techniques such as rinsing, ascending migration or sedimentation and percoll gradient separation. In situations where only single gametes are available, rinsing and spinning of the ejaculate sample is sufficient. In case of obstructive azoospermia, sperm are obtained by microsurgical epididymal sperm aspiration (MESA) or testicular sperm aspiration (TESA). Oocytes collected by ovarian puncture are subjected to short-term hyaluronidase activity to remove granular cells that form the cumulus oophorus and the corona radiata. This allows for an accurate assessment of the maturity of the testes and the ooplasmic state.
Oocytes in the second meiotic division (metaphase II) show the presence of the first polar body in the perivitelline space, and can be directly subjected to microinjection. Eggs with a slight degree of immaturity, in the first meiotic division (metaphase I), after a short cultivation (6-12 hours) usually achieve full in vitro maturation. Oocytes in the first meiotic division do not fully mature in vitro and are not fertilized. In the essential part of the procedure sperm with the most favourable construction and mobility is identified, it is then immobilised by mechanical damage to its tail, and introduced into the interior of the injection micropipette. The holding pipette positions the oocyte in such a way that the polar body is at 12 o’clock.
In this position, the insertion of the injection micropipette takes place at 3 o’clock due to the position of the carokinetic spindle of the second meiotic division, usually located under the polar body. Immediately following the penetration of the zona pellucida, the ooplasm is aspirated into the pipette, which enables the oolemma to be interrupted and the sperm is deposited in the ooplasm. The slow removal of the pipette should allow the oolemma repair process to begin. About 9-12 hours after the procedure it is usually possible to assess egg activation and the process of fertilization. Subsequent procedures are similar to those of the classic in vitro method and include observation of embryo development, selection of embryos with the greatest developmental potential and transfer to the uterus, as well as cryopreservation of excess embryos and luteal phase supplementation.
TESA / PESA
TESA / PESA
Sometimes the semen contains no sperm at all. It is important then to answer the question: do men’s testes produce sperm?
Preliminary analysis of hormone levels and assessment of the size and consistency of the testes are helpful. In most cases genetic diagnostics of both the man and the woman are necessary to assess the risk of genetic disorders in the offspring. If the results are positive, we will perform a fine needle biopsy of the testes or epididymis. It is performed under general anaesthesia, so it is painless and less burdensome for the man. He will be able to leave the clinic about an hour after the test. If the biopsy material contains live sperm, fertilization is possible by microinjection into the egg, as described above. In each case, the sperm obtained from the testes or epididymides are frozen for later use. In the absence of live sperm, the choices available are obtaining sperm from a donor, or adoption.
Intracytoplasmic Morphologically Selected Sperm Injection is a cytoplasmic injection of sperm, morphologically selected by means of high magnification and high resolution techniques. The IMSI method is based on the ICSI method, differing by the way sperm is selected for micromanipulation.
The effectiveness of IVF treatment, although very high, is still unsatisfactory in some patient groups. Therefore, new methods are being used worldwide that may have a beneficial effect on clinical outcomes. The use of non-invasive, risk-free solutions to improve performance is ideal. The IMSI method is the best example.
In order to increase the chances of fertilization during infertility treatment, preparative methods are being used increasingly more often to obtain the best quality sperm fractions. This is especially important when performing the ICSI procedure. Selection of sperm is made on the basis of their construction and motility. However, in some cases of male infertility when using normal technical equipment for ICSI, choosing the right sperm by the embryologist may be impeded. The IMSI performed before injecting the sperm into the egg, allows to view the sperm head (core) under very large magnification, which makes selection easier.
The use of IMSI also improves the chances in cases of weak ovarian response to stimulation. Obtaining a small number of oocytes poses the risk that after their fertilization we will not get a satisfactory number of normal embryos. The IMSI method of choosing the best sperm improves the chances of obtaining normal embryos, and hence the chance of pregnancy.
Clinical studies confirm that using the IMSI method improves the pregnancy rate and reduce the rate of miscarriages. This procedure is especially recommended for couples with previous ICSI failure, severe male infertility factor, and who have a history of miscarriages.
The following pictures show a comparison of sperm images with ICSI and IMSI. Thanks to the large magnification in the IMSI method, we can accurately assess the morphological structure of the sperm and select the ideal one.
It is a device that allows constant monitoring of embryo development from the moment of fertilization of the oocyte until it is transferred to the uterus or cryopreservation. The ovaries collected during ovarian puncture are fertilized in an embryo laboratory and then placed in suitable culture media and inserted into a CO2 incubator. Temperature, humidity and composition of gases provide optimum conditions for embryo development. The task of the embryologist is to evaluate the rate of embryonic division, to determine the number and symmetry of dividing cells (blastomeres), the degree of early embryo fragmentation, and the development of the embryo in the fifth embryogenesis stage (blastocyst). It should be emphasised that embryonic development is a very dynamic process and its changes are very rapid. For this reason, the usual one-time evaluation of the embryo under the microscope during 24 hours of culture does not allow to monitor for many key developmental details that determine the developmental potential of the embryo. A more frequent assessment consisting of removing incubated embryos and observing them under a microscope may adversely affect its development due to unstable culture conditions.
At present it is possible to continuously and automatically monitor and document the development of embryos without having to remove them from the incubator. This is possible thanks to the time-lapse technique. The embryoscope is currently the most advanced system for cultivation and continuous monitoring of embryo development. Inside the device is a camera that automatically captures the embryos every few minutes. The images obtained are part of a detailed film of the entire embryonic development process. The analysis of the resulting film facilitates the selection of the embryos with the best division rate and morphology, and thus having the greatest development potential. It also allows you to identify abnormal embryos. Thanks to this method it is possible to reduce the number of embryos transferred to the uterus without reducing the effectiveness of the treatment and also avoiding the risk of multiple pregnancies. Normally developing embryos that have not been transferred to the uterus are frozen for later use in future cycles. It is possible to analyse the film with the patients, which helps them to understand the problems with the effectiveness of the treatment.
This method can be used by all couples, but is especially recommended for older women and those who experienced repeated IVF failure. Our experience shows that using this method contributes to a significantly better outcome.
Incision of the embryo’s zona pellucida
Incision of the embryo’s zona pellucida
Successful implantation or implantation of the embryo in the endometrium depends partially on the smooth process of its hatching from the zona pellucida (ZP). This allows direct contact of the blastocyst (embryo at the developmental stage capable of implantation) with the endometrium. The blastocyst expands and presses from the inside into the ZP, which breaks and allows the blastocyst to escape it. This phenomenon is called blastocyst hatching. Sometimes the ZP does not break, which prevents the interaction of the embryo with the endometrium and thus the implantation and development of pregnancy. Scientific studies and clinical observations show that this is caused by morphological factors, such as: the average thickness of the ZP, embryo fragmentation, or the insufficient number of blastomeres, which make up the embryo. Other factors that may hinder the blastocyst hatching process are likely to be the age of the patient and the associated zona hardening. It has also been found that the freezing and thawing of embryos may be the cause of this problem.
Assisted hatching is a laboratory technique that involves the thinning or cutting of the ZP surrounding the embryo. This treatment makes it easier for the blastocyst to hatch, thereby increasing the chance of successful implantation in the endometrium. It is done just before the transfer – this applies to both fresh and frozen embryos. The risk of damaging the embryo is very small. However, the data suggest an increased incidence of twin pregnancies after this procedure.
Indications for this procedure include:
Indications for this procedure include:
Women with elevated FSH levels in 1-3 days of the cycle
Morphology of the embryo: e.g. too thick ZP, delayed cell division or high percentage of cellular fragmentation
In cycles after previous failed IVF treatments
Before the cryotransfer
Evaluation of uterine contractions
Evaluation of uterine contractions
Transfer of embryos into the uterus is the final stage of the IVF treatment. This short procedure is the culmination of the time-consuming preparations, sometimes difficult stimulation, ovarian punctures, and then the entire embryonic process. Successful implantation of embryos after transfer is mainly affected by embryo quality and uterine receptivity or “the desire of the womb to accept them”. Equally important are the quality of the procedure and the conditions for the embryo. The key component of implantation may be excessive (more than 3 contractions/3 minutes) uterine contractions. Increased uterine contractions, both spontaneous and traumatic, may reduce the chance of IVF-ET embryo implantation, resulting in the removal of embryos from the uterus after transfer.
Excessive uterine contraction is present in approximately one-third of patients treated with IVF-ET. The results of treatment (pregnancy rate) achieved in this group of patients are even 3 times lower in comparison to patients with normal activity. As it is known, uterine contractions are conditioned by the stimulation of oxytocin receptors, which creates the possibility of pharmacological intervention. It has been shown that the use of oxytocin receptor antagonists (Atosiban) reduces the contraction of the uterus and improves embryo implantation. It has also been shown that Atosiban has no embryotoxic effects and its administration during transfer is safe. Preliminary clinical findings from the study of this drug indicate an increase in the pregnancy rates in patients treated with IVF-ET. After a period of conducting preliminary studies at our centre, we introduced this procedure as a routine procedure for patients diagnosed with excessive uterine contractions.
Planning intrauterine insemination should be based on a proven patency and proper functioning of the fallopian tubes and the fertility of the semen. In case of ovulation disorders, the specific treatment can include a pharmacological induction of ovulation in combination with natural procreation. The effectiveness of this procedure is difficult to assess due to the low homogeneity of the groups and the difficulty in objectifying the research findings. However, it is assumed that it does not exceed 7-20% of pregnancies per treatment cycle.
When the partner’s reproductive potential is only slightly decreased, the method of choice is intrauterine insemination (IUI), often in combination with ovulation induction. The purpose of the procedure is to increase the number of competent male gametes at the site of syngamy. For this purpose, the semen is inserted into the vagina, most often with the use of a cervical cap, which allows for long-term contact of the semen with the cervical canal, and the more intense penetration of the sperm into the upper part of the reproductive organs. The more advanced procedure is intrauterine insemination, where properly prepared sperm are introduced into the uterine cavity. This allows you to use the entire pool of live sperm from the ejaculate and avoid losses typical for natural reproduction.
The most well-known of these procedures is intraperitoneal insemination, where relevant fractions of normal sperm are isolated using swim-up techniques, as where Percoll gradient separation occurs, found in the in vitro lab. Such prepared sperm are introduced into the fallopian tubes or peritoneal cavity of the female, into the immediate vicinity of the ova. These methods are effective in 14-60% of pregnancies per treatment cycle, and depend mainly on the reproductive potential of the partner, provided that the semen is properly qualified. The ineffectiveness of the above procedures used during the 6 cycles of treatment should lead to reconsideration of indications, and application of more advanced methods of in vitro fertilization.
Insemination with the sperm of the husband or donor
This method requires a one-day medical visit during the first three days of menstruation. We evaluate the initial situation, and establish indications and contraindications to treatment. If ovulation induction is required, we determine its optimum method, the scope of monitoring and the expected date. Based on the partner/donor semen analysis, we determine the technical details of the procedure (cervical, intrauterine, intratubal insemination, etc.).
A gynaecologic ultrasound examination is a non-invasive screening test to evaluate a woman’s reproductive system. Thanks to the use of ultrasonic waves generated by advanced ultrasound devices, and the use of state-of-the-art technical solutions in the software of the device, it is possible to obtain very precise images of individual structures.
A gynaecological ultrasound examination aims to assess the following:
uterine walls – including the uterus (detection of developmental defects of the uterus), the presence of pathological structures (myoma/adenomyosis lesions, tumours);
endometrium – its condition and the presence of endometrial polyps, endometrial hyperplasia, endometrial cancer, as well as assessment of the endometrial function (for hormone disorders, cycle monitoring)
ovaries – assessment of the structure (presence of tumours, cysts) and ovarian function (follicular apparatus, presence of ovarian follicle), and evaluation of ovarian stimulation for IVF needs
fallopian tubes – proper functioning fallopian tubes cannot be examined by the ultrasound test, but diagnostics of their pathology is possible, such as the oviduct hydrocephalus, ectopic pregnancy, and ovarian cysts
The Douglas cavity – exclusion/confirmation of the presence of fluid in the cavity, as well as pathologies (tumours, endometriosis)
The gynaecologic ultrasound is most often performed with a transvaginal probe (in sexually active patients), while in other cases it may be performed using an abdominal or rectal probe.
The use of 3D ultrasound technology has made it possible to circumvent many of the limitations of 2D projection. 3D technology allows you to save a very large series of sequential ultrasound images into a volumetric file that can be archived and processed later. The transvaginal 3D ultrasound allows simultaneous visualisation of a given organ in three perpendicular planes for accurate measurements of dimensions and volumes, while simultaneously viewing the external contours of the examined structure. The saved volumetric image can be rotated and viewed in all planes. In reproductive medicine, 3D ultrasonography has many potential applications, including assessment of congenital and acquired uterine defects, ovarian patency, ovarian observation and their response to ovulation stimulation, as well as the evaluation of endometrial receptivity. In addition, the three-dimensional Power Doppler angiography allows you to assess the flow volume in the ovary and endometrium.
Indications for ultrasound examination:
Periodic assessment of the reproductive system (prophylactically every 2 years)
Diagnostics of developmental disorders of the reproductive system
Diagnostics of uterine pathology, appendages
Early pregnancy diagnostics
Diagnostics of gynaecological endocrinological disorders
Computer Assisted Semen Analysis (CASA)
Computer Assisted Semen Analysis (CASA)
Semen analysis is a basic component of infertility diagnostics. The parameters taken into account for the assessment of semen in accordance with the guidelines of the World Health Organization (along with the 2010 reference values) are:
Reference values for semen analysis according to WHO 2010 guidelines:
Routine semen analysis was performed using an optical microscope. Unfortunately, this is a subjective assessment, and though it does not make it difficult to assess the sperm count, there may often be deviations when assessing sperm motility. The inability to objectively analyse the percentage of progressive sperm has resulted in the development of computer assisted semen analysis (CASA). This system consists of combining phase contrast microscopy with a camera, computer and specialised software, which allows to view subsequent sperm positions at a frequency of 5-60 images per second, and transfer the saved images into digital form. It is subjected to a computer analysis allowing for accurate and objective analysis of the motility trajectory of each sperm.
Female hormonal teksts
Hormonal tests should be performed in cases of suspected endocrinopathy, and in all patients starting IVF treatment. They should include: folliculotropin (FSH), luteinizing hormone (LH), estradiol, prolactin (in specific cases), and progesterone in the middle of the second phase of cycle.
Chronic anovulation is diagnosed when the concentration of progesterone, measured at least three times in the middle of the luteal phase, is less than 3 ng/ml. To evaluate the ovarian reserve, it is important to determine the FSH level during days 2-5 of the cycle.
If it is elevated, diagnostics can be expanded to include the AMH (Anti-Müllerian hormone) and inhibin B tests. It is not currently recommended to routinely test prolactin levels in patients with regular menstrual periods. However, it should be performed if the menstrual cycle is irregular. Prolactin concentrations in excess of 200 μg/l (9 nmol/l) are an indication for further diagnostics due to the risk of pituitary adenoma.
Hormonal tests are also very useful for assessing the presence and quality of ovulation. It is highly probable that ovulation cycles are present in a woman who menstruates regularly every 21 to 35 days. This is confirmed by an ultrasonographic examination showing the presence of a growing follicle reaching a diameter of 16-22 mm in the periovulatory phase, and determining oestradiol concentrations in the periovulatory phase ( 150 – 300pg/ml), as well as serum progesterone seven days before expected menstruation (above 10ng/ml).
Endocrine diagnostic tests are also useful in determining indications for, plans and monitoring of treatment. FSH values above 10-12 mIU/ml in women indicate a reduced ovarian reserve and a high probability of a poor response to stimulation, while its values above 20mIU/ml indicate ovarian insufficiency. Additional determination of AMH and inhibin B values allows for more accurate evaluation of reproductive potential, but also to optimise the work of medications used for induction of ovulation, and reduces the rate of failed stimulations and complications. This mainly concerns the stimulation and induction of ovulation in patients undergoing medically assisted reproduction.
Male hormonal tests
The basic indications for hormone evaluation are azoospermia (lack of sperm in the ejaculate) and oligozoospermia: <5 million sperm in 1 ml of semen. The basic diagnostics should include the measurement of serum FSH and serum testosterone levels. For hypoandrogenic symptoms, the level of inhibins, LH, estradiol and prolactin should be tested in addition to FSH levels.
Elevated FSH values indicate spermatogenic disorders. This is not a clear-cut study: elevated FSH levels have been observed with spermatogenesis, and normal values were present with spermatogenic disorders. If azoospermia is detected in the semen sample and the andrological examination is normal, a biopsy of the testes is required. Histopathological examination of the content of the testis is aimed at finding the preserved course of spermatogenesis and the presence of spermatozoa.
The best predictor of the presence of spermatozoa in the testicles or epididymis is the serum FSH concentration test. If FSH> 15 mjm./ml, the chance of their presence in the tested content is <20%, if FSH is > 25 mjm./ml it is close to zero. Therefore, the serum FSH test can be considered as one of the stages of differential diagnosis of azoospermia (due to obstruction of the seminal tract) resulting in the presence of normal FSH values. With non-obstructive azoospermia, FSH values may be elevated or normal, and testosterone levels may be lower. Reduced FSH, LH and testosterone levels are observed in hypogonadotropic hypogonadism.
It is a minimally invasive method of gynaecological endoscopy. According to the scope of the examination and indications for its implementation, either diagnostic or surgical hysteroscopy may be performed.
Diagnostic hysteroscopy – is a particularly important test in the diagnosis of uterine endometrial disorders. By using a hysteroscope, a special optical device equipped with an optical fibre, it is possible to accurately assess the cervical canal and the uterine cavity.
When examining the uterine cavity, the focus is on the following:
Presence of abnormalities in the cavity structure – diagnosis of uterine defects (bicornuate uterus, uterine septum)
Presence of pathology within the endometrium – presence of endometrial polyps, intrauterine adhesions, endometrial hyperplasia
Presence of submucosal fibroids
Reactive processes of the fallopian tubes
Indications for diagnostic hysteroscopy include infertility, repeated failed IVF treatment, miscarriages, and abnormal uterine bleeding. Diagnostic hysteroscopy is usually performed in the first half of the cycle (days 7-13 of the cycle) by inserting a thin hysteroscope (4-5 mm) through the vagina and cervix into the uterine cavity. Thanks to the presence of the camera it is possible to record the procedure and to back the examination results with photographic documentation. Diagnostic hysteroscopy can be performed without anaesthesia because, due to the very small diameter of the hysteroscope, it is not a painful procedure. However, if the patient experiences discomfort or pain, a short-term intravenous anaesthetic may be administered by the anaesthetist.
Surgical hysteroscopy – is used to treat disorders detected during diagnostic hysteroscopy. The hysteroscope is equipped with a tube through which surgical tools are inserted to remove the lesions – endometrial polyps, fibroids, adhesions, septum or hyperplasia. The following measures must be taken prior to undergoing the surgical hysteroscopy procedure:
Patient should be fasting (for at least 6 hours)
Patient should be vaccinated against hepatitis B
Current lab test results:
Coagulation tests APTT and PT
Serological tests for chlamydia
HIV, HCV, HBsAg, VDRL, anti-HCV (in the past 6 months)
Degree of purity
It is a test to diagnose abnormalities of:
Structure of the uterus (submucosal fibroids, congenital defects of the uterus, septum)
The procedure is performed on a gynaecological chair and requires the insertion of a speculum. After disinfecting the cervix, a sterile balloon catheter is inserted into the cervical canal, filled with fluid in order to secure it. Next, a saline solution or special substance is administered into the catheter. Liquid fills the uterine cavity. While the liquid is being administered a transvaginal ultrasound is performed. The shape and size of the uterine cavity, and the presence of pathological endometrial bulges are evaluated. The patency of the fallopian tubes is checked by evaluating the passage of fluid from the uterine cavity through the fallopian tubes to the abdominal cavity. After the test, the catheter is removed from the uterus and the patient can return to daily activities. The treatment usually lasts no longer than 30 minutes.
Preparing for the test:
The test is performed in the first week after menstruation (after bleeding and spotting have completely stopped)
Previously performed detailed gynaecological ultrasound examination (preferably with documentation)
Evaluation of vaginal purity (vaginal microbiological smear)
Current cytology (performed within one year)
Treatment for men with a spinal cord injury
Treatment for men with a spinal cord injury
There are situations where the problem lies with the man and is due to inability to obtain semen. This is often the case in men who have suffered a permanent spinal cord injury in the past and are now wheelchair-bound. In such cases, the semen can be obtained surgically through a biopsy of the epididymis or testes (MESA/TESA). In cases where the obtained semen shows normal values in terms of number and quality of sperm, insemination is sufficient. Unfortunately, it often does not meet the required norm, having very short sperm viability and a significant percentage of dead sperm, which is an indication for ICSI IVF treatment. As in other cases, once obtained, the reproductive material of a man can be frozen and used repeatedly.
Since infertility causes can be found in both men and women diagnostics should be conducted jointly and simultaneously in both partners. Among the most common causes of infertility in women are ovulatory disorders. In situations where it is manifested by lack of menstruation, identifying the problem is relatively simple. More often, however, the problem is caused by qualitative disorders, which result in lowering the reproductive potential of the egg. In some cases the treatment is quite simple and effective, other times it proves difficult and expensive, and in some patients it is not possible at all.
The second cause of infertility concerns the male partner and the ability of his semen to fertilize the egg. At this point it should be emphasized that measuring the quality of the semen is subject to the highest percentage of false positives, which often results in wrongly qualifying for treatment. Hence the significance of a reliable and objective examination of the semen.
The third most common cause of infertility concerns the anatomy and function of reproductive organs. A healthy, normal state of the fallopian tubes, uterus and neighbouring organs is a prerequisite for fertilization, followed by the implantation of the embryo as well as its proper development in the uterus. Typical disorders in this area include obstruction of the fallopian tubes, abdominal adhesions, endometriosis or uterine fibroids.
Properly conducted diagnostics must address each of the potential causes listed above, which are relevant to the planned treatment. There is no need to investigate the patency of the fallopian tubes, as due to the male factor being responsible, treatment includes extracorporeal fertilisation, etc. In every case, however, the success of the treatment requires accurate identification of the causes of infertility and the biological determinants of reproductive efficiency.
At Kriobank, basic infertility diagnostics include a medical history interview complete with analysis of previous treatment, gynaecological examination, semen analysis, and assessment of the prevalence of ovulation with an attempt to determine its quality. The diagnostic process takes into account the specificity of the clinical situation of each couple, and is intended to provide information on the individual reproductive potential of the woman and the man, as well as their joint reproductive potential. This type of treatment oriented diagnostics makes it possible to determine further action.
Accurate determination of the reproductive potential requires the assessment of the concentrations of basic hormones that control the female, and sometimes male, reproductiveness. In every case we conduct detailed examination of the semen. In order to ensure the safety of patients and future pregnancies, it is necessary to evaluate their infectivity and possibility of carriage of certain diseases, in accordance with international standards. In certain cases we test the patency and function of the fallopian tubes (HSG), assess the state of the reproductive system and its environment (laparoscopy), and the state of the uterine cavity (hysteroscopy). In cases where repeated attempts at treatment have failed, we assess the functional status of the uterus using advanced testing methods.
Jesteśmy realizatorem projektu Bon „antywirusowy” w ramach Programu Operacyjnego Województwa Podlaskiego na lata 2014-2020. Osi Priorytetowej I Wzmocnienie potencjału i konkurencyjności gospodarki regionu, Działania 1.4 Promocja przedsiębiorczości oraz podniesienie atrakcyjności inwestycyjnej województwa, Poddziałania 1.4.2 Podniesienie atrakcyjności inwestycyjnej BOF, Typ projektu: Bon „antywirusowy” – projekt grantowy. Pozyskaliśmy dofinansowanie na zakup środków ochrony indywidualnej i zbiorowej, które wykorzystywane są w prowadzonej działalności i funkcjonowaniu placówki medycznej o zaostrzonym reżimie sanitarnym.