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Print Posted on 07/19/2017 in Integrative Understanding of Infertility

Female Infertility: Causes, Testing, Treatment

Female Infertility: Causes, Testing, Treatment

INTRODUCTION

At present, the issues of researching the essence of female’s infertility: its causes, testing and treatment have become an interdisciplinary research mainstream focus for both paradigms of modern Gynecology and reproductive endocrinology. Female infertility has determined scientists’ very close attention to the key issues “female infertility and its causes;” “female infertility and testing” “female infertility and treatment”, which can be vividly illustrated by the fact that recently there have been more than 53000 analytical overviews concerning this question published (just according to PubMed data) let alone numerous scientific publications and research papers all over the world. Together with this, despite the problem of female infertility has been inclusively described and analyzed comprehensively in modern reproductive sciences, we have chosen the major causes and revealed the basic information about them.

Should a couple have trouble conceiving, despite trying for more than a year, both partners may be checked for issues leading to infertility. In 45% – 50% of infertile couples, the problem is solely with the female partner. Infertility in a woman may be the only reason that a couple can’t conceive, or it may simply add to the difficulties caused by infertility in his partner.

Female infertility, its causes, testing and treatment, is a really complicated topic. Though the following is general information, which represents basic causes of a female’s infertility, we strongly recommend that you consult a specialist in female reproductive health who can fully evaluate, diagnose and treat your particular situation.

(1)           FEMALE REPRODUCTIVE CELLS AND EMBRYO IMPLANTATION PROCESS

The ovary is a ductless reproductive gland which form part of the female reproductive system and in which the female reproductive cells are produced. Females have a pair of ovaries, held by a membrane beside the uterus on each side of the lower abdomen. They are oval in shape, about four centimeters long and lie on either side of the womb (uterus) against the wall of the pelvis in a region known as the ovarian fossa. They are held in place by ligaments attached to the womb but are not directly attached to the rest of the female reproductive tract, e.g. the fallopian tubes. The ovaries have two main reproductive functions in the female body. They produce oocytes (eggs) for fertilization and they produce the reproductive hormones, estrogen and progesterone. The function of the ovaries is controlled by gonadotrophin–releasing hormone released from nerve cells in the hypothalamus which send their messages to the pituitary gland to produce luteinizing hormone and follicle stimulating hormone. These are carried in the bloodstream to control the menstrual cycle. The ovaries release an egg (oocyte) at the midway point of each menstrual cycle. Usually, only a single oocyte from one ovary is released during each menstrual cycle, with each ovary taking an alternate turn in releasing an egg.

The most excessively wonderful thing to know about new born baby girls is that all of them are born with immature egg cell (oocytes) in her ovaries, nestled in fluid–filled cavities called follicles. Between 16 and 20 weeks of pregnancy, the ovaries of a female fetus contain six to seven million oocytes. At 20 weeks, a female fetus has a fully developed reproductive system, replete with six to seven million eggs. Most of the oocytes gradually waste away, leaving about 1 to 2 million presents at birth. None develop after birth. At puberty, only about 300,000–400,000 more than enough for a lifetime of fertility remain; and approximately one thousand eggs are destined to die each month. This phenomenon is completely independent of any hormone production, birth control pills or pregnancies. Only a small percentage of oocytes mature into eggs. The many thousands of oocytes that do not mature degenerate. Degeneration progresses more rapidly in the 10 to 15 years before menopause. All are gone by menopause. Only about 400 eggs are released during a women’s reproductive life, usually one during each menstrual cycle will be released from the ovary (in a process called ovulation) and be capable of being fertilized in the fallopian tubes/uterine tube/oviduct of the female reproductive tract. Of the roughly 400 follicles that will reach ovulation over the course of a woman’s fertility, a woman can expect 20 follicles to mature each month with just one egg being released. Until released, an egg remains dormant in its follicle–suspended in the middle of cell division. Thus, the egg is one of the longest–lived cells in the body. Each egg carries half a potential baby’s genetic information. (The other half, of course, comes from sperm.)

In the ovary, all eggs are initially enclosed in a single layer of cells known as a follicle, which supports the egg. Over time, these eggs begin to mature so that one is released from the ovary in each menstrual cycle. As the eggs mature, the cells in the follicle rapidly divide and the follicle becomes progressively larger. Many follicles lose the ability to function during this process, which can take several months, but one dominates in each menstrual cycle and the egg it contains is released at ovulation. As the follicles develop, they produce the hormone estrogen. Once the egg has been released at ovulation, the empty follicle that is left in the ovary is called the corpus luteum. This then releases the hormones progesterone (in a higher amount) and estrogen (in a lower amount). These hormones prepare the lining of the uterus for potential pregnancy (in the event of the released egg being fertilized). If the released egg is not fertilized and pregnancy does not occur during a menstrual cycle, the corpus luteum breaks down and the secretion of estrogen and progesterone stops. Because these hormones are no longer present, the lining of the womb starts to fall away and is removed from the body through menstruation. After menstruation, another cycle begins.

Egg quantity (ovarian reserve) and egg quantity

The term ‘ovarian reserve’ refers to a female’s current supply of eggs [the number of eggs still left in the ovary] and is closely associated with reproductive potential. Each month, during a female’s reproductive cycle, a single follicle is selected out of a group of potential follicles, reaches maturity, and ovulates a single egg. 

It is absolutely essential to understand that the concept of egg quantity is vital for conceiving. Previously, there was the accepted scientific view that women are born with a fixed number of eggs she will ever have, and as she ages, the number becomes smaller and smaller and that the body has no capacity to increase this supply. While the number of eggs decreases with age, that does not mean that younger women are in the clear. Some women are born with fewer eggs, making them have a low egg quantity at a much earlier age.

The less eggs a woman has, the harder it is for her hormones to recruit and develop an egg. With irregular ovulation, it can be very difficult for a woman to become pregnant, and with no ovulation, it is impossible for a woman to become pregnant on her own. Nowadays, an exclusive discovery challenges notion that women are born with a fixed number of eggs, but the female ovary may be able to grow new eggs in adulthood, and if confirmed, the discovery would overturn the previous scientific theory.

What is vital to consider when the concept of Female Egg Quality is discussed that it is absolutely synonymous with the probability of embryo implantation. The concept of a woman’s “Egg Quality” is derived from the observation that the probability of embryo implantation is strongly related to the age of the woman who provides the egg and to her ovarian reserve. That is why, Female “Egg Quality” is synonymous with “the probability of embryo implantation”. Female Egg Quality can’t be determined by glancing at the egg, measuring its receptivity to fertilization by sperm, or observing initial embryo division. Just because an embryo looks perfect in the laboratory, does not mean that it will implant. The only proof of Female Egg Quality is in the embryo implantation.

Embryo implantation process

Implantation is the final frontier to embryogenesis and successful pregnancy. The embryo enters the uterine cavity approximately 4 to 5 days post fertilization. After passing down the fallopian tube or an embryo transfer catheter, the embryo is moved within the uterine lumen by rhythmic myometrial contractions until it can physically attach itself to the endometrial epithelium. It hatches from the zona pellucida within 1 to 2 days after entering the cavity thereby exposing the trophoblastic cells of the trophectoderm to the uterine epithelium. Implantation occurs 6 or 7 days after fertilization. During implantation and placentation, a human embryo must attach itself to the uterus under conditions of shear stress. The embryo is rolling about within a mucus rich environment between the opposing surfaces of the endometrial walls of the uterus. This interactive process is a complex series of events that can be divided into three phases: apposition, attachment and invasion [6].

Apposition phase of the embryo implantation process: once the blastocyst hatches from the zona pellucida, the free–floating sphere of cells must orient itself as it approaches the endometrial surface and form an initial adhesion (apposition) before it can firmly attach and begin the process of invasion. The apposition stage is a transient and dynamic process whereby the embryo “tethers” itself to the endometrial surface. Uterine contractions and mucin secretion within the uterine cavity propel the blastocyst around the cavity. Despite these fluid dynamics which creates shear stress, the embryo is able to approach the wall, roll around to right itself so that the trophectoderm overlying the inner cell mass apposes to the endometrial surface. During this apposition phase, there is a dialogue between the floating blastocyst and endometrium using soluble mediators such as cytokines and chemokines acting in a bidirectional fashion to guide the blastocyst onto a 3–dimensional “docking” structure. The hormone–regulated pinopodes at the endometrial surface have been shown to mark the timing and appearance of optimal endometrial receptivity [1].

Embryo attachment phase of the embryo implantation process: shortly after the apposition, integrin–dependent adhesion and attachment occurs. This receptor mediated event allows the blastocyst to attach firmly to the uterine wall and trophoblasts transmigrate across the luminal epithelium, burying the embryo beneath the uterine wall. Integrins are a family of cell adhesion molecules present on the plasma membrane as heterodimeric and glycoprotein subunits. These cell surface receptors on both the trophoblasts and the endometrium are involved at multiple functional levels during implantation. The trophoblast integrins have been shown to mediate cell–cell and cell–matrix interaction in trophoblast attachment, migration, differentiation and apoptosis. On the endometrial surface, most of the integrins serve housekeeping roles, but there are three heterodimers whose expression marks the boundaries of the implantation window [5]. The embryo regulates these integrins in the endometrium at the protein level in the apposition phase [7]. Therefore, after the blastocyst tethers to and breaks through the glycocalix barrier, it then induces by paracrine crosstalk a favorable epithelial integrin pattern for its firm attachment.

Trophoblast Invasion phase of the embryo implantation process: the final phase of embryo implantation is invasion which permits the creation of the hemochorial placenta. The medical term invasiveness implies the ability of cells to cross anatomic barriers. The first barrier is the layer of endometrial epithelial cells upon which the trophoblasts are attached. Immediately beneath the epithelial layer of cells is a specialized type of matrix called the basement membrane, a thin continuous layer composed mainly of type IV collagen. The basement membrane functions as an anchor for surface epithelium and a separating layer by ensheathing blood vessels, muscle cells and nervous tissue. Beyond the basement membrane lies a highly variable matrix, called the interstitial stroma which contains other tissue cells, vessels and lymphatic channels. Since the blastocyst is too large to squeeze through the epithelial layer, the attached trophoblasts use paracrine activity to induce an apoptotic reaction in the underlying endometrial epithelial cells [3].

To achieve successful invasion, trophoblasts then differentiate in a tightly regulated manner producing anchoring cytotrophoblasts and highly secretory synciotrophoblasts that degrades the extracellular matrix with several proteases. The expression of matrix metalloprotease – 9 coincides with the peak invasive potential of trophoblasts. By the 10th day after fertilization, the blastocyst is completely embedded in the stromal tissue of the endometrium with the epithelium regrown over to cover the site of implantation. Eventually, the cytotrophoblasts invade the entire endometrium, the uterine vasculature and into the inner third of the myometrium. The access to the uterine vessels creates the lacunar networks comprising the uteroplacental circulation which places the placental trophoblasts in direct contact with maternal blood. The placenta then serves its vital role as the transfer organ between mother and fetus for nutrients, gas and waste products, hormones and growth factors, among others.

Improper invasion can lead to compromised placental development and complications of pregnancy. Where there is excessive invasion, a placenta may attach onto the myometrium (accreta), into the myometrium (increta) and completely through the myometrium (percreta). Each of these is associated with higher risks of hemorrhage, operative delivery and hysterectomy. If invasion is too shallow, it may lead to intrauterine growth restriction and/or preeclampsia [8].

Fertilized egg’s fate: why some embryos don’t implant?

There are some of the noteworthy contributing factors to embryo implantation failure are Diminished Ovarian Reserve, Advanced Maternal Age and Diminished Egg Quality.

Diminished Ovarian Reserve

Ovarian reserve refers to the reproductive potential left within a woman’s two ovaries based on number and quality of eggs. Diminished ovarian reserve is the loss of normal reproductive potential in the ovaries due to a lower count or quality of the remaining eggs. Diminished ovarian reserve (DOR) occurs when a woman’s ovaries lose their reproductive potential, which can cause infertility. One of the major conditions leading to infertility in women, diminished ovarian reserve (DOR) is characterized by a low number of eggs in a woman’s ovaries and/or impaired development of the existing eggs. A woman’s ovarian reserve refers to the quality and quantity of her eggs, and diminished ovarian reserve means those factors are decreasing. A woman who has elevated follicle–stimulating hormone (FSH) levels on the third day of menses is said to have diminished “ovarian reserve”. What this means physiologically is that the ovary is producing less feedback signal to the pituitary gland, and the body responds by making more follicle–stimulating hormone (FSH) in an effort to stimulate the ovary. It has been observed over the past five years in thousands of cycles of fertility treatment that women with elevated follicle–stimulating hormone (FSH) levels have markedly decreased Egg Quality and rarely conceive with their own eggs (they do, however, become pregnant readily with donor eggs). The precise physiological reason for this is unclear. However, we do know that when eggs are obtained from women with elevated follicle–stimulating hormone (FSH) levels, they appear normal, they fertilize normally, and undergo initial embryonic cleavage at a normal rate. However, they rarely divide beyond the eight–cell stage and almost never implant.

Advanced Maternal Age

Even if follicle–stimulating hormone (FSH) levels are normal, the age of the woman providing the eggs plays a major role in determining Egg Quality. Just as with women with elevated follicle–stimulating hormone (FSH) levels, eggs obtained from women in their late 40’s appear normal, fertilize normally, and undergo initial embryonic cleavage in a normal manner. However, such embryos almost never implant. Because of low implantation rates in women over 45 years old, normal follicle–stimulating hormone (FSH) levels are not considered “reassuring”.

Diminished Egg Quality

Another way in which age may impact egg quality is through what’s called the battery effect. In the reproductive process, there’s about a week between the time an egg is fertilized and the time it implants in the uterus. During that week, the mitochondria in the egg act as batteries to power the fertilized egg through the process of cellular division or meiosis. Researchers have noted that while the eggs of older women may appear of good quality and have little trouble getting fertilized, they often stop dividing at some point between fertilization and implantation. The mitochondria in older eggs may lose the “battery power” to complete this stage of the process.

The egg is connected to the circulation prior to ovulation, and it is connected again after embryo implantation. But during the seven days between ovulation and implantation, the egg and the embryo which results from it are contained within the zona pellucida and are dependent on energy coming from the mitochondria which were in the egg at the moment of ovulation (no mitochondrial replication takes place until after implantation). The older egg usually looks normal at the time of ovulation and its initial fertilization and embryonic development remain normal. This is because its energy stores are still adequate. However, it soon runs out of batteries and stops dividing. Implantation is not achieved because the embryo stops dividing before it reaches the implantation stage. The scientists do not yet know how to increase the energy stores to an egg prior to ovulation. When Egg Quality is low, the only therapy that has a proven track record and produces reliable results is egg donation.

Another way in which age may impact egg quality is through what’s called the battery effect. Just as batteries in your various devices wear out over time, so too may the “batteries” that power an egg. In the reproductive process, there’s about a week between the time an egg is fertilized and the time it implants in the uterus. During that week, the mitochondria in the egg act as batteries to power the fertilized egg through the process of cellular division or meiosis. Researchers have noted that while the eggs of older women may appear of good quality and have little trouble getting fertilized, they often stop dividing at some point between fertilization and implantation. The mitochondria in older eggs may lose the “battery power” to complete this stage of the process.

(2)           THE MOST COMMON CAUSES OF FEMALE INFERTILITY: OVARIAN DYSFUNCTION

The most common cause of infertility is the failure to ovulate, which affects about 40–50% of women with infertility. A failure to ovulate can result from:

Primary Ovary Insufficiency (POI) (also called Primary Ovarian Failure or premature menopause) is a failure of the ovary to function adequately in a woman younger than 40 years, in its role either as an endocrine organ or as a reproductive organ; it is also a condition characterized by amenorrhea, hypoestrogenism, and elevated serum gonadotropin levels [in women younger than 40 years]. In primary ovarian insufficiency, ovaries do not regularly release eggs and do not produce enough sex hormones despite high levels of circulating gonadotropins (especially follicle–stimulating hormone [FSH]).

Predictable menstrual cyclicity represents the healthy ovarian function during the reproductive years. Each month, highly coordinated hormonal and ovarian morphological changes develop and release a mature oocyte that is ready for fertilization. A disruption of this process may result in anovulation and ovarian steroid hormone deficiency. An autoimmune disorder is common and causes the body to attack ovarian tissues, resulting in the premature loss of eggs and an inability to produce eggs. It can be due to genetic abnormalities or damage from chemotherapy and radiation. It is also caused by thyroid, adrenal and hormone disorders.

Polycystic Ovary Syndrome (PCOS), also known as polycystic ovarian syndrome caused by an imbalance of reproductive hormones and is a common cause of female infertility. Women with polycystic ovarian syndrome (PCOS) have abnormalities in the metabolism of androgens and estrogen and in the control of androgen production. Polycystic ovarian syndrome (PCOS) can result from abnormal function of the hypothalamic–pituitary–ovarian (HPO) axis. It is a condition that involves changes in the hypothalamus, pituitary and ovaries which results in a hormonal imbalance that negatively affects ovulation. The hormonal imbalance creates problems in the ovaries. The ovaries make the egg that is released each month as part of a healthy menstrual cycle. With PCOS, the egg may not develop as it should or it may not be released during ovulation as it should be. It can cause problems with menstrual cyclicity and make it difficult to get pregnant.

Fallopian Tube Disease

The fallopian tubes are two thin tubes, one on each side of the uterus, which help lead the mature egg from the ovaries to the uterus. When an obstruction prevents the egg from traveling down the tube, a woman has a blocked fallopian tube, also known as tubal factor infertility. This can occur on one or both sides and is the cause of infertility in 40 percent of infertile women.

Fallopian tubal disease is a disorder in which the fallopian tubes are blocked or damaged. Fallopian tubal disease affects about 25% of infertile women. Blockage of the tubes may be due to adhesions or complete blockage. Blocked or damaged fallopian tubes restrict the egg [if the fallopian tubes are damaged, the egg may fail to reach the uterus to be fertilized] and subsequent embryo from making it from the ovary into the uterus, thereby causing infertility. In addition, some women are born with blockages in their fallopian tubes.

One main cause of tubal disease is scar tissue (adhesions), resulting from endometriosis, gynecological surgery, bowel surgery, Cesarean section, ruptured appendix or other internal trauma. Sexually Transmitted Diseases, which go undetected or untreated, such as chlamydia or gonorrhea, can damage the fallopian tubes irreparably. Tubal ligation (having your tubes tied to prevent pregnancy) and/or subsequent reversal of tubal ligation can also leave your fallopian tubes damaged.

If the fallopian tubes have adhesions or scar tissue around them, it can block an egg and subsequent embryo from reaching the uterus, causing infertility. If the tubes are partially blocked by adhesions, sperm may meet the egg in the fallopian tube instead of in the uterus, and an ectopic pregnancy may occur.

(3)           THE MOST COMMON CAUSES OF FEMALE INFERTILITY: DISEASES AND CONDITIONS OF THE UTERUS

At least 10% of female infertility is caused by an abnormal uterus.

Endometriosis is defined as the presence of endometrial–like tissue outside the uterus, which induces a chronic, inflammatory reaction [4]. The exact prevalence of endometriosis is unknown but estimates range from 2 to 10% within the general female population but up to 50% in infertile women [2]. With increasing severity of endometriosis, scar tissue (adhesions) becomes more common and the chance of natural conception decreases. Treatment options include pain relievers, hormones, and laparoscopic excision surgery for endometriosis.

Endometriosis occurs when the cells that normally line the uterine cavity, called endometrium, grow outside the uterus instead. Every month a woman’s body goes through hormonal changes. Hormones are naturally released which cause the lining of the womb to increase in preparation for a fertilized egg. If pregnancy does not occur, this lining will break down and bleed – this is then released from the body as a period. Endometriosis cells react in the same way – except that they are located outside the womb. During the monthly cycle hormones stimulate the endometriosis, causing it to grow, then break down and bleed. This internal bleeding, unlike a period, has no way of leaving the body. This leads to inflammation, pain, and the formation of scar tissue (adhesions). Endometrial tissue can also be found in the ovary, where it can form cysts, called ‘chocolate cysts’ because of their appearance. Endometriosis is most commonly found inside the pelvis, around the ovaries, the fallopian tubes, on the outside of the womb or the ligaments (which hold the womb in place).

There are four basic scientific theories for why minimal to mild endometriosis causes infertility:

– Problems with egg transport down the fallopian tube;

– Failure of the egg sac (follicle) to release its egg (luteinized unruptured follicle syndrome);

– Toxins in peritoneal fluid (naturally occurring fluid within the body cavity);

– An abnormal immune response (antibodies).

Why does endometriosis cause infertility?

Scar tissue (adhesions) is similar to cobwebs and can be fine, or dense. Adhesions are more common in moderate and severe endometriosis. This scar tissue distorts the pelvic anatomy. If the ovary is wrapped in adhesions, the released egg gets trapped and is unable to reach the tube. The tubes and ovaries dangle down in another important area called the Pouch of Douglas. If this pocket is covered by adhesions, then the chance of getting pregnant is also lower. Research has found a link between infertility and endometriosis. Studies show that between 25% and 50% of infertile woman have endometriosis, and between 30% and 40% of women with endometriosis are infertile. Anatomical distortion and adhesions caused by endometriosis, particularly in moderate and severe disease, reduces the chance of natural conception. The chance of conceiving with minimal to mild endometriosis is not very different from normal. The more severe the endometriosis, the smaller the chance of getting pregnant naturally. This is because there are more adhesions that trap the egg and prevent it from getting down the Fallopian tube.

Uterine Fibroids are growths that appear within and around the wall of the uterus. Fibroids can arise in different positions in the uterus. Most women with fibroids do not have problems with fertility and can get pregnant, but if they cause significant distortion of the cavity of the uterus they may increase the risk of miscarriage. Fibroids may reduce the possibility of conception if they distort the fallopian tube as it enters the womb or if they distort the cavity of the womb thus reducing the chance of implantation. Fibroids often contribute to infertility and are found in 5% to 10% of infertile women.

Uterine Polyps are growths of tissue that form a mass in a woman’s uterus and they can cause infertility. Uterine polyps are soft red outgrowths from the lining of the womb (the endometrium), usually less than 1 cm in diameter, which often flatten to fit the cavity of the uterus. Polyps are either flat (sessile) or pedunculated, meaning they have a stalk. Most uterine polyps are endometrial polyps that form on the endometrium, which is the lining of the uterus. Uterine polyps are usually noncancerous, but they may develop into cancer. Endometrial polyps can cause infertility, though how polyps decrease fertility has not been proven. They may inhibit sperm movement or prevent successful embryo implantation in the uterus. The presence of the hormone estrogen appears to promote the growth of uterine polyps. Surgical removal of the polyps can increase the chances for a woman to get pregnant.

Uterine scarring from previous injuries or surgery. Intrauterine adhesions, also known as synechiae or scar tissue, are bands of fibrous scar tissue that form within the uterus. Scar tissue affects the functional lining of the uterus and can be a reason for infertility.

Woman’s uterus has three layers: an outer layer (serosa), a middle muscular layer (myometrium), and an inner layer (endometrium). The inner layer contains the lining that sheds once a month with a woman’s menses. It is also the place where an embryo implants and grows into a pregnancy.

Uterine scarring decreases the ability to get pregnant because it decreases the blood supply to the endometrial lining. It may also cause the cavity to be completely scarred. A healthy endometrium is important for an embryo to implant. Scar tissue can also be a physical barrier for sperm to enter the upper uterus to fertilize an ovulated egg.

Uterine scarring is caused by trauma to the lining with a procedure and/or inflammation. Potential causes for scarring include: (1) Surgical: Dilation and curettage for prior abortion, miscarriage, retained placenta after delivery, abnormal uterine bleeding; Cesarean section; Myomectomy; (2) Inflammation/infection: Endometritis – infection of uterine cavity; Other infections (Chlamydia, Tuberculosis, Schistosomiasis).

Uterine scarring decreases the ability to get pregnant because it decreases the blood supply to the endometrial lining. Uterine scarring may increase the risk of miscarriage and infertility. It may also cause the cavity to be completely scarred. A healthy endometrium is important for an embryo to implant. Scar tissue can also be a physical barrier for sperm to enter the upper uterus to fertilize an ovulated egg.

Congenital defects that cause an unusually shaped uterus can affect a woman’s ability to remain pregnant after conception.

Chronic infections in the cervix can also reduce the amount or quality of cervical mucus, the sticky or slippery substance that collects on the cervix and in the vagina. Reduced amount or quality of cervical mucus can make it difficult for women to get pregnant.

(4)           FEMALE INFERTILITY TESTING 

Ovulation dysfunction

Approximately 15% of couples will experience infertility due to ovulation dysfunction – the woman not consistently releasing an egg each month. Many tests can be used to document ovulation.

Menstrual History – menstrual history alone can often determine whether a woman is ovulating. Women who ovulate generally have regular cycles with consistent amounts of blood flow each month. A woman who has irregular bleeding, frequent bleeding, wide variation in the amount of bleeding, or long episodes without any bleeding at all is likely to have an ovulation disorder.

Hormonal Assessment – all women should be screened with a cycle day 3 FSH (Follicle Stimulating Hormone) and estrogen level. This simple test will indicate whether or not ovarian function may be diminished. In women with amenorrhea (no menses) a very high FSH may indicate premature menopause or premature ovarian failure. Women over 35 need further assessment of their ovarian reserve with a clomiphene citrate challenge test or an antral follicle count by ultrasound and a blood test for AMH (Anti– Müllerian Hormone). In addition, a simple test of the thyroid gland (TSH), as a screening of thyroid function and a serum prolactin should be obtained. It is recommended that women with excessive bodily hair growth have blood testing for male hormone levels, such as testosterone. All women normally produce these hormones, but excessive production may be associated with abnormal ovulation.

Progesterone level – Progesterone is a hormone that the ovary produces after a woman ovulates. A blood progesterone level greater than 3 ng/ml is a very accurate indicator that a woman has ovulated that cycle.

Basal Body Temperature Charting (BBT) – a fertility specialist may ask you to keep a BBT chart to determine if you are ovulating. Generally, after a woman ovulates the release of progesterone causes a mid–cycle temperature rises of ½ to 1 degree Fahrenheit. Thus, a woman who is ovulating will have a biphasic temperature chart – lower temperatures before she ovulates and a rise in temperature after she ovulates. The rise in temperature only occurs after ovulation, so a temperature chart cannot be used to time intercourse for conception. Some women may be ovulating even though the temperature chart is unclear, and factors such as a cold or the timing of taking one’s temperature can affect the accuracy of this testing.

Ovulation predictor kits – Many over–the–counter ovulation predictor kits can be used to determine if a woman is ovulating. These urine kits detect the surge of luteinizing hormone (LH) that occurs just before a woman ovulates. We highly recommend the use of these kits since many procedures we perform require accurate timing, and these kits are one way to detect the day of ovulation.

Ultrasound – a fertility specialist may elect to perform an ultrasound examination to look for developing egg follicles on your ovaries, which may be an indication of impending ovulation. If a follicle on the ovary appears collapsed, this may be an indication the ovulation has occurred. Ultrasound has evolved into the most important modality to monitor one’s ovulary process.

Ovarian reserve tests

Ovarian reserve testing is the process of evaluating several hormone levels between days 2, 3, and 4 of a woman’s menstrual cycle to determine the quantity of eggs that she has available. Ovarian reserve tests were developed by IVF clinics to predict how a woman having IVF treatment would respond to the drugs used to stimulate the ovaries and ultimately how many eggs she may produce. Ovarian reserve can be assessed through blood tests to measure three important hormones: estrogen (E2), follicle stimulating hormone (FSH) and anti–Müllerian hormone (AMH) or by an ultrasound scan that counts the growing follicles within each ovary.

Follicle stimulating hormone (FSH) is produced by the brain and travels through the blood stream to the ovaries where it stimulates the growth of follicles. FSH is a hormone that stimulates ovarian follicles (each follicle has the potential to release an egg) and is highly predictive of fertility. A normal FSH level is somewhere between 2–8.9: this is enough to support the growth of one follicle and is how nature normally limits us to singleton pregnancies. As follicle stimulating hormone (FSH) levels vary through the cycle it must be measured in the first few days of menstruation (day 2–5 of the cycle). While elevated follicle stimulating hormone (FSH) levels mostly affect women in their late thirties and early forties, a younger woman with an elevated follicle stimulating hormone (FSH) level also has a reduced likelihood of establishing a pregnancy without the use of donor eggs.

As a homodimeric glycoprotein of the transforming growth factor-β (TGF- β) superfamily, anti–Müllerian hormone (AMH) is emerging as an important regulator of follicle development. Produced by granulosa cells of growing follicles after birth, anti–Müllerian hormone (AMH) levels normally are low in primary follicles, increase to maximal levels in large preantral and small antral stages, and then decline during final follicular maturation, becoming restricted to cumulus cells surrounding the oocyte. Serum anti–Müllerian hormone (AMH) concentrations correlate with the numbers of antral follicles before and with the ovarian response to gonadotropin therapy for IVF–ET, and diminish after oophorectomy to those of ovary–intact postmenopausal women. Within the mammalian ovary, moreover, anti–Müllerian hormone (AMH) inhibits follicle recruitment and FSH–dependent follicle growth as well as selection, thereby suppressing aromatase activity during early folliculogenesis.

AMH is a protein made by the cells that surround each egg. The more eggs a woman has, the higher her AMH level. A simple blood test can determine AMH levels. AMH levels can be drawn at any point in the menstrual cycle and results generally do not vary cycle–to–cycle. Ideally, AMH levels should be greater than 1.2 ng/ml. Levels less than 1.2 ng/ml are concerning and might lead to earlier or more aggressive fertility treatment to maximize the likelihood of a successful pregnancy. Low AMH levels are associated with a low likelihood of success.

In contrast, anti–Müllerian hormone (AMH) is produced by the growing follicles and is a direct marker of the number of follicles. Anti–Müllerian hormone (AMH) varies with age but normal levels are somewhere between 3 and 35. Lower levels are indicative of poor reserve and higher levels associated with, but not diagnostic of, polycystic ovaries. Anti–Müllerian hormone (AMH) varies less through the cycle and so can be measured at any time.

Luteinizing hormone (LH) is a hormone produced by the pituitary glands in the brain. It works with follicle-stimulating hormone (FSH), which another gonadotropin made in the pituitary gland. FSH stimulates the ovarian follicle, causing an egg to grow. It also triggers the production of estrogen in the follicle. The rise in estrogen tells your pituitary gland to stop producing FSH and to start making more LH. The shift to LH causes the egg to be released from the ovary, a process called ovulation. In the empty follicle, cells proliferate, turning it into a corpus luteum. This structure releases progesterone, a hormone necessary to maintain pregnancy. If fertilization occurs, luteinizing hormone will stimulate the corpus luteum, producing progesterone to sustain the pregnancy. If pregnancy doesn’t occur, the levels of progesterone drop off and the cycle begins again. An increased amount of Luteinizing hormone (LH) is released when ovulation is about to occur. This increase allows the egg to mature within a follicle and sets ovulation in motion.

Another method to assess ovarian reserve and the chance for pregnancy is by assessing the woman’s antral follicles, which are small follicles that can be measured with ultrasound. An antral follicle count is a good predictor of the number of follicles in the woman’s ovaries that may produce a mature egg.

The growing follicles can be seen on ultrasound. During the scan, which has to be an internal scan because the follicles are so small, the follicles are counted and you are given a total antral follicle count (AFC). The total antral follicle count (AFC) and Müllerian hormone (AMH) are related therefore: some clinics measure one and not the other whilst some clinics measure both. Müllerian hormone (AMH) and total antral follicle count (AFC), which may provide some information about egg quality as well as egg number, are better markers of ovarian reserve than FSH. However, a raised FSH level (≥9) is important as it does correlate with a poorer response to ovarian stimulation and is used by many CCGs to determine funding. It is important to consider that poor result from ovarian reserve testing does not signify an absolute inability to conceive and should not be the only criteria considered to limit or deny access to infertility treatment.

Clomid Challenge Test

The Clomid Challenge Test (also known as the clomiphene citrate challenge test, or CCCT) is a blood test used to assess ovarian reserve. Ovarian reserve is indicative of the quality and quantity of a woman’s eggs. During this test, the woman’s Follicle Stimulating Hormone (FSH) level is taken on day two, three, or four of her menstrual cycle. FSH is responsible for stimulating the eggs in the ovary to begin maturation. On days five through nine she takes the fertility medication Clomid (clomiphene citrate). On day 10 of the cycle, FSH levels are obtained to assess the patient’s ovarian reserve. Blood tests are analyzed in our state-of-the-art, Joint Commission accredited, endocrine laboratory adjacent to our King of Prussia office.

Abnormalities of the Uterus and Fallopian Tubes

Hysterosalpingogram (HSG) – an X–ray procedure done by a fertility specialist, generally during the first half of your menstrual cycle. It involves placing dye into your uterus that will show up on X-rays. The dye outlines the inside of the uterus and fallopian tubes. If the fallopian tubes are open, the dye passes into the surrounding pelvic cavity and is reabsorbed by your body. You may experience some cramping with this test which generally disappears quickly. You will be instructed to take over–the–counter pain medication (acetaminophen, ibuprofen) before the test to help with cramping. You will also be given antibiotics to take prior to the procedure.

Sonohysterogram – This test involves performing a transvaginal ultrasound during the introduction of sterile saline solution into the uterus through the cervix. This test will help define the shape of the uterine cavity and can identify uterine abnormalities. It generally cannot be used to identify problems with the fallopian tubes.

Cervical Factor: Abnormal Sperm/Mucus Interaction

Post–Coital Test – this test is performed around the time of your expected day of ovulation, usually determined by the ovulation predictor kit. You will be asked to have intercourse with your partner (without lubricants) the night before or the day of your appointment. You may shower but should not tub bathe or douche. Your doctor will take a small sample of mucus from your cervix (no different from a Pap smear) to be examined for the presence of motile sperm. A transvaginal ultrasound will be done at the time of the post-coital test.

Blood Tests

Fertility problems sometimes originate from hormone irregularities. Other tests can be important to do prior to becoming pregnant. Blood tests include: Thyroid testing, Rubella titer, Blood type and antibody screen, AMH level (a test of your ovarian reserve), Baseline FSH and estradiol levels, Hemoglobin A1C (a diabetes screening test), Infectious disease tests.

Hysteroscopy

A procedure where a telescope–like instrument is inserted through the cervical canal into the uterine cavity, for the evaluation of the interior of the uterus. It is an important procedure because it allows for diagnosis and treatment of small surface lesions inside the uterine cavity (e.g., polyps, scarring or adhesions) that adversely affect the ability of an embryo to attach to the uterine lining. Such lesions are often missed through the performance of an HSG. Commencing at least 17 days before the expected next menstrual period (i.e., usually about 10 days following the initiation of menstruation), urine should be collected twice daily and tested for the onset of the spontaneous LH surge. The initiation of the LH surge usually precedes ovulation by 8 to 36 hours. In order to detect the onset of the LH surge as early as possible, it is important that urine be tested at least twice daily. This is done as follows: the bladder is emptied first thing in the morning, upon awakening. One half-hour later urine is collected (only a very small amount is required) and tested using an over-the-counter LH kit (obtainable at a drug store). The earliest sign of any color change should be documented. It need not be a pronounced color change as suggested by the insert in the kit. Any alteration in coloration is significant. The same process of testing is then repeated at night before going to sleep.

Laparoscopy

A procedure where a telescope–like instrument is introduced through the belly button into the abdominal/pelvic cavity allowing diagnosis and treatment of ovarian cysts/endometriomas/benign tumors, uterine fibroids, tubal blockage, ectopic pregnancy, appendicitis, pelvic adhesions, etc. Laparoscopy is usually performed as an out-patient procedure with the patient under general anesthesia. It is one of the only ways to diagnose early pelvic endometriosis accurately.

Endometrial Biopsy

This is a simple in–clinic procedure, whereby a sliver of uterine lining (endometrium) is removed and sent to the laboratory to evaluate histologic changes in the endometrium.

Sclerotherapy for Ovarian Cysts (Endometriomas)

Sclerotherapy is a safe and effective alternative to surgery for definitive treatment of recurrent ovarian endometriomas in a select group of patients planning to undergo In Vitro Fertilization (IVF). Sclerotherapy for ovarian endometriomas involves: needle aspiration of the liquid content of the endometriotic cyst, followed by the injection of 4–5% tetracycline into the cyst cavity. Treatment results in disappearance of the lesion within 6–8 weeks in more than 75% of cases so treated. Ovarian sclerotherapy can be performed under local anesthesia or under general anesthesia. It has the advantage of being an ambulatory office–based procedure, at low cost, with a low incidence of significant post–procedural pain or complications and the avoidance of the need for laparoscopy or laparotomy.

CONCLUSION

FEMALE INFERTILITY TREATMENT OPTIONS AND ADDITIONAL PROCEDURES

The purpose for all of this testing is to arrive at a treatment plan that can improve fertility. More than 50% of women will have a treatable cause of female factor infertility.

Oocytes Retrieval: as part of the In Vitro Fertilization (IVF) process, oocytes are retrieved from the woman’s ovaries for fertilization – a process known as oocyte (egg) retrieval. Following light sedation of the woman, an ultrasound machine is used to visualize, locate and retrieve individual eggs from the ovarian follicles. The samples (follicular aspirates) are passed directly from the operating room to the embryology laboratory, where they are examined for eggs. As eggs are located under a microscope, the embryologist isolates them and places them in a holding dish, where they are separated from the oocyte cumulus cells that surround them. The isolated eggs are then washed and placed into an incubator, with patient–specific identifiers on the dish and the incubator. The next phase in processing is determined based on the patients’ individual treatment protocol. Options include: Insemination (Fertilization); Oocyte (Egg) Freezing; Intracytoplasmic Sperm Injection (ICSI).

IVF (In Vitro Fertilization): in practice, female’s eggs and male’s sperm meet in specially designed plastic test tubes or dishes, and this is where fertilization will take place. It takes another three to five days for embryos to develop from the fertilized eggs. Once this has occurred, the laboratory team confers with the couples’ physician as to the quality of the embryos, and the selection as to which one(s) is made based on the lab findings as well as the couples’ wishes. The embryo transfer is an atraumatic procedure to place one or more embryos into the womb of the female partner. Ultrasound is frequently employed to assist the physician in accurately placing the embryos in an optimal spot for implantation of the embryos in the uterine lining. This procedure requires no sedation and takes about five minutes to perform. A blood test about 12 days later will determine if pregnancy has taken place.

Sometimes patients need additional IVF (In Vitro Fertilization) laboratory procedures to improve (1) the chances of successful fertilization, (2) successful “take” of the transferred embryos, and (3) identification of embryos that have either a normal complement of chromosomes, or will not result in a pregnancy that carry a specific genetic mutation. 

The IVF Laboratory can assist in (1) by a procedure called intracytoplasmic sperm injection, or ICSI.  Improving the chances of implantation (2 above) by transferred embryos in certain patient categories can be accomplished by a technique called assisted hatching (AH). Chromosomal or genetic analysis of preimplantation embryos (3 above [Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic Screening]) is perfect opportunity to have a baby (babies) free of inherited disease(s).

ICSI (Intracytoplasmic Sperm Injection): the intracytoplasmic sperm injection (ICSI) is a procedure involves injecting sperm into a mature egg to assist in the fertilization process. First, the embryologist prepares a sterile ICSI dish with a microdrop of polyvinylpyrrolidone (PVP), a viscous media designed to slow sperm motility so they are easier to locate before injection into the egg. In the same dish, the embryologist also prepares multiple drops of standard micromanipulation media so multiple eggs can be prepared. A protective cover of oil is then applied to keep the media warm and prevent the drops from evaporating. After that, the embryologist completes a micromanipulation setup by aligning a holding pipette and ICSY needle with the microscope. An ICSI dish is prepared and labeled, and the sperm sample and eggs are both double–verified against the patients’ paperwork. Using a microscope and two 3D joysticks, the embryologist then places a small sample of the processed sperm in the PVP drop and moves the eggs to the dish. The embryologist uses the ICSY needle and pipette to isolate and immobilize the normal–looking sperm before depositing it into the individual egg. This process is repeated with every egg in the sample. The eggs are then placed back into their original media drops, and the dish is returned to the incubator.

Embryo Biopsy for Genetic Analysis: the endometrium is the inside layer of the uterus where the embryo embeds and continues its development. The uterus must be free of obstructions such as fibroids or significant congenital abnormalities. Endometrial cells have the capacity to rapidly grow and develop under the influence of the hormones estrogen and progesterone. This “growth” results in the increased vascularity and endometrial width which is needed to support a developing embryo. A small sample of the endometrium is taken during the luteal phase of the ovulatory cycle approximately twelve days after the LH surge. The biopsy is examined to see if the cellular development of the endometrium “matches” what is predicted for day the sample was taken. If endometrial cellular development matches the biopsy, the biopsy is termed “in phase”. If the biopsy shows less than predicted development for that cycle day, the biopsy is said to be “out of phase” and a luteal phase defect may exist. Progesterone is administered in the next ovulatory cycle to correct the luteal phase defect.

Egg Freezing and Storage: before beginning the oocyte (egg) freeze, the embryologist prepares a dish with several drops of vitrification media for each egg to be frozen. Next, the patient’s paperwork and samples are verified for accuracy, at which point the prepared eggs are removed from the incubator and transferred to the prepared dish. After placing any remaining eggs back in the incubator, the embryologist moves the eggs through the various drops of vitrification media in a carefully timed process. The eggs are then loaded onto a storage device and plunged into liquid nitrogen to complete the vitrification process.

Egg Thawing: embryo thawing is the procedure to recover previously frozen embryos prior to an embryo transfer. After the embryo is retrieved from liquid nitrogen storage and all patient identifiers are checked, it is placed in a prepared dish and processed through several drops of thaw materials. The dish is then moved to a heated surface to complete the warming process. Once thawed, the embryo is moved into a new dish to be washed in culture media, documented and transferred to the incubator to await the embryo transfer procedure.

Embryo Transfer: the embryo transfer procedure involves implanting embryos into the woman’s uterus. Prior to the procedure, the embryos are placed into a special transfer media for equilibration in the incubator. The actual procedure takes place in the embryo transfer room, which is connected to the laboratory to minimize the time the embryos are outside the incubator. Following a multi–step, double–witnessed verification of the woman’s identity (including the embryos and embryo dishes), the embryos are prepared in the laboratory and placed into a transfer catheter. Next, the woman is lightly sedated, and the embryologist places the transfer catheter into the uterus under ultrasound guidance.

Insemination: during the insemination process, the sperm and eggs are combined. Before the procedure is performed, both the embryologist and a lab technician check the eggs and sperm against the patient’s paperwork. The embryologist then injects a small amount of the selected sperm from the semen sample into the drops on the sample dish. At this point, the embryologist examines the sample dish under the microscope and on a video screen to ensure the sperm are swimming normally.

Assisted Hatching: assisted hatching is a micromanipulative ART procedure designed to help embryos “hatch out” of their “’shell”, the zona pellucida, and implant into the endometrium. Once the embryos mature and are ready for transfer, the embryologist gently makes a small hole in the zona pellucida. This opening can be made with a microscopic needle, chemically using Acid Tyrode’s solution, or with a laser. The assisted hatching procedure does not damage the embryo. It is often used in older females, those who have failed previous IVF cycles, and when poor embryo quality or abnormally thickened zona pellucidae are present.

Embryo Check Days 1, 3 and 5

Day 1 Fertilization Check: the first fertilization check occurs in the morning to evaluate whether eggs have properly fertilized over the evening to become embryos. The individual oocytes (eggs) are isolated and placed into a petri dish to be washed in the embryo culture media, then examined under a large, inverted microscope. Once the embryologist has determined if the eggs have matured into zygotes, (s)he places the dish back in the incubator and documents the results.

Day 3 Embryo Check with Grading: as part of the day 3 embryo evaluation, the embryologist reviews the embryos under a microscope, taking video documentation along the way. In addition to supplementing the patient’s paperwork, this information is used to complete a fertilization and embryo report for the patient’s physicians. As part of this process, a grading system is used to rate embryos as good, fair or poor based on their cell quantity and quality.

Day 5 Embryo Check with Blastocyst Grading: on the fifth day, the embryologist examines the blastocysts under a microscope and takes pictures to be included with the patient documentation. Once the embryos are returned to the incubator, the embryologist records his or her findings, a process that includes classifying each embryo within a pre-determined framework based on its growth, development and cell quality. This rigorous evaluation process allows us to apply a uniform grading scale within the lab and affords patients greater insight into the development of their embryos.

Time Lapse Imaging – The Embryoscope

The Embryoscope device comprises an incubator, a microscope and a computer operating system. This innovative tool allows the embryologist to monitor every embryo at ten–minute intervals in seven different video cuts, for a 3D, time–lapse view of each embryo’s development.

The Embryoscope is a time–lapse monitoring incubator with built–in cameras, which takes pictures of the embryos every 10 minutes and compiles videos of embryo development. This means that the embryologists never have to remove the embryos from the incubator until they are chosen for embryo transfer. Standard practices for culturing embryos that do not use the Embryoscope, involve removing embryos from the incubator at 3 to 5 time points, in order to assess embryo development. By doing so, the embryos are exposed to drops in temperature and a change in the environment. these types of checks have to be done very quickly, which does not leave much time to gain enough information about embryo development. The Embryoscope allows the embryologists to constantly monitor embryo development without causing stress to the embryo. The embryologists can therefore access the maximum amount of information regarding development and the likelihood of success.

REFERENCES

[1] Bentin–Ley U., Sjogren A., Nilsson L., Hamberger L., Larsen J.F., Horn T. Relevance of endometrial pinopodes for human blastocyst implantation. Hum Reprod Suppl., 2000; Vol. 6, pp. 67–73.

[2] Eskenazi B., Warner M.L. Epidemiology of endometriosis. Obstet. Gynecol. Clin. North. Am., 1997; 24:235–258.

[3] Galan A., Herrer R., Remohi J., Pellicer A., Simon C. Embryonic regulation of endometrial epithelial apoptosis during human implantation. Hum Reprod., 2000; Vol.15 (suppl. 6), pp. 74–80.

[4] Kennedy S., Bergqvist A., Chapron C., D’Hooghe T., Dunselman G., Greb R., Hummelshoj L., Prentice A., Saridogan et al. ESHRE guideline for the diagnosis and treatment of endometriosis. Hum. Reprod., 2005; 20:2698–2704.

[5] Lessey B.A. Adhesion molecules and implantation. J Reprod. Immunol., 2002, Vol. 55, pp. 101–112.

[6] Norwitz E., Schust D., Fisher S. Implantation and the survival of early pregnancy. NEJM, 2001; Vol. 342,19, pp. 1400–1408.

[7] Simon C., Gimeno M.J., Mercader A., O’Connor J.E., Remohi J., Polan M.L., Pellicer A. (1997). Embryonic regulation of integrins beta 3, alpha 4, and alpha 1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab, Vol. 82, pp. 2607–2616.

[8] Zhou Y., Damsky C., Fisher S. (1997). Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype: one cause of defective endovascular invasion in this syndrome? J Clin Invest. Vol.99, pp. 2152–2164.

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