Fertility Analysis Revision PDF

FERTILITY ANALYSIS Dr : Amira Madany Revision The hypothalamic-pituitary-testes axis Endocrinopathies and male reproduction. (A) Neuroendocrine regulation by hypothalamic– pituitary–gonadal (HPG) axis maintains the normal secretion and functions of reproductive hormones....

FERTILITY ANALYSIS Dr : Amira Madany Revision The hypothalamic-pituitary-testes axis Endocrinopathies and male reproduction. (A) Neuroendocrine regulation by hypothalamic– pituitary–gonadal (HPG) axis maintains the normal secretion and functions of reproductive hormones. Gonadotropin-releasing hormone (GnRH) is synthesized by the hypothalamus, which stimulates the anterior pituitary to secrete the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Whereas, gonadotropin-inhibitory hormone (GnIH) inhibits the anterior pituitary gonadotropin synthesis and release. In Leydig cells, LH acts to aid steroidogenesis. FSH acts on the Sertoli cells, supporting spermatogenesis. Sertoli cells secrete activin and inhibin among other substances, which mediate positive and negative feedback on the HPG axis, respectively.  The Leydig cells mainly generate testosterone, which is secreted in a pulsatile manner and binds to serum proteins like albumin and sex hormone–binding globulin (SHBG). Leydig cells also produce smaller amounts of estradiol and dihydrotestosterone (DHT) by aromatase. Testosterone is the primary regulator of LH, and estradiol is the primary regulator of FSH. ▪ Prolactin is a polypeptide hormone that is responsible for lactation, breast development. It secreted from anterior pituitary gland. ▪ It release has an inhibitory effect on the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. ▪ The loss of GnRH results in a lack of pulsatile stimulation of gonadotrophic cells resulting in the loss of FSH and LH release from the anterior pituitary. ▪ Similarly, prolactin in males inhibits GnRH release resulting in decreased spermatogenesis and infertility, but this is considered pathologic. ▪ In males, serum prolactin levels range from 2 to 18 ng/ml, in females 2 to 30 ng/ml, and during the third trimester of pregnancy, 10 to 210 ng/ml. Inhibin B ▪ Inhibins are glycoproteins belonging to TGF-β family of proteins. ▪ It is produced by the Sertoli cells in response to FSH stimulation. Inhibin B in turn controls FSH through negative feedback mechanism. It is the marker of Sertoli cell function. ▪ Inhibin suppresses GnRH-stimulated release of FSH. Epididymal Functions Sperm Transport: from rete testes to vas deferens. Sperm Concentration: through absorption of 90% of the fluid that leaves the rete testis. Sperm Protection: Epididymal epithelial cells excrete various antioxidant enzymes, including superoxide dismutase, into the epididymal lumen to neutralize reactive oxygen species and prevent sperm cell damage. a blood-epididymis barrier functions to shield maturing sperm cells from the immune system and from harmful substances that might exist in the bloodstream.  Seminal vesicles 1. Produce 60% of alkaline semen rich with fructose., which is yellowish and viscous slightly. 2. Produce prostaglandins stimulate muscular contractions within the female reproductive organs. These contractions aid the movement of sperm cells toward the egg cell. 3. The prostaglandins have a role in immune modulation. They regulate the pathways that may exacerbate inflammation in the female reproductive tract during physiological processes such as ovulation, implantation, and parturition, e.g., ejaculation or the spermatozoa induce an inflammatory response in the endometrium in the preimplantation period after mating. 4. Produce the major clotting proteins, Semenogelins (I, II), help the semen to become sticky and jelly-like after ejaculation (they immediately form a coagulum that entraps spermatozoa upon ejaculation). 5. Produce fibrinogen, facilitates in seminal clotting after ejaculation. This clotting of the semen helps to keep the ejaculated sperms in the female reproductive tract. 6. The seminal fluid also includes ascorbic acid, inorganic phosphorus and potassium. 7. The fluid from the seminal glands also contains citric acid and a coagulating enzyme known as vesiculase.  Prostate contributes 20%of semen , a milky acidic fluid containing: 1. Acid phosphatase (acid phosphomonoesterase ) for activation of sperm, 2. Citric acid and zinc (nourishment). Citric acid its principle role is to maintain pH, it plays a vital part in sperm motility and hyaluronidase activity. Zinc affects the stability of sperm chromatin and it play a regulatory role in the process of capacitation and the acrosome reaction. Zinc is the major inhibitor of the prostatic KLK proteolytic cascade, the triggering cues of which depend on ejaculatory stimuli driven by the central nervous system.  Prostate 1. After ejaculation, within a few minutes the activated KLKs, including PSA, start the semen liquefaction process that enables sperm to be released and move towards the Fallopian tube. 2. Kallikreins (KLKs), a specific subfamily of 15 serine proteases, which include PSA 3. Prostate-specific antigen (PSA), playing a key role in enabling the sperm to swim into the uterus by keeping the semen in liquid form by cleaving semenogelin (I and II) in the seminal coagulum. 4. Milky semen filters and removes toxins for protection of the sperm.  Cowper's (bulbourethral) gland: produce 20% of mucoid alkaline semen rich with which galactose, sialic acid and mucin, Play a role in: 1. mucoprotein-rich fluid that helps to lubricate the distal urethra and neutralize acidic urine that has remained in the urethra. 2. lubricating the urethra and external urethral orifice to protect sperm from mechanical damage during ejaculation. 3. allowing more efficient sperm transfer.  Common causes of male infertility 1. Varicocele: swelling of the veins that drain the testicle. Or an abnormal collection of bulging veins above the testicle; accounting for 38% of cases. (decreased no. of sperms, decreased motility and abnormal morphology).  Common causes of male infertility 4. Undescended testicle (Cryptorchidism): It is an inability of testes to descend down into scrotum, reported in about 2 to 6% newborns. 5. Hormonal imbalance : This hormonal imbalance may be caused by genetic condition, gland malfunction (hypothalamus, pituitary, thyroid and adrenal glands)and unhealthy weight; these factors can interfere with the production of sperm and ultimately can influence male fertility. Any fluctuation in luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone can affect spermatogenesis in males. Evaluation of Male Infertility ❑Reproductive History ❑Medical History ❑Physical Examination ❑Semen analysis ❑Endocrine Evaluation ❑Advanced analysis  Semen analysis Semen analysis is one of the most important investigations in the assessment of male fertility potential. Semen analysis provides a global measure of ✓ testicular and epididymal function (for sperm production and maturation, respectively), ✓ vasal patency (for sperm transport), and ✓ accessory sexual gland function (for production and delivery of seminal plasma). Macroscopic Evaluation of Semen Physical examination (color, volume, PH, liquefaction and Viscosity o Semen color: grayish white Possible causes, by color, include: Red semen: This could be caused by inflammation of the prostate or the glands that help produce semen. Yellow or green semen: This could be caused by an infection, jaundice, or the presence of vitamins or medication in the semen. Macroscopic Evaluation of Semen Physical examination (color, volume, PH, liquefaction and Viscosity o The normal volume of ejaculate after 2–7 days of sexual abstinence ranges from 2 to 6 ml. o However, there are other possibilities as follows: 1. Aspermia: No ejaculate after orgasm. 2. Hypospermia: Less than 0.5 ml of semen. This can be due to improper collection, hypogonadism, partial retrograde ejaculation, and congenital bilateral absence of the vas deferens (CBAVD), and obstruction of lower urinary tract may yield low volume. 3. Hyperspermia: More than 6 ml of semen. This can be attributed to prolonged abstinence or excessive secretion from the accessory sex glands and also occurs in cases of male accessory gland infection.  Semen PH: Alkaline 7.2- 8.2 ▪ The main component of semen is a coagulated alkaline fluid that comes from the seminal vesicles. ▪ This fluid along with the sperm from the vas deferens empties through the ejaculatory duct. Prostatic fluid, the second largest component of seminal volume, generally has a relatively acidic pH of 6.5 and combines with the seminal fluid and sperm in the urethra. Prostatic fluid does not traverse the ejaculatory ducts.  Semen PH: Alkaline ▪ Normal semen pH is in the range of 7.2–8.2 and it tends to increase with time after ejaculation. ▪ Changes in PH of semen are usually due to inflammation of the prostate or seminal vesicles. ▪ A low volume sample with measured pH below 7.0 indicates obstruction of the ejaculatory ducts. Microscopic Sperm Analysis Sperm Concentration Sperm count is typically reported as concentration (millions of sperm per milliliter). (15 million per ml) Total sperm count (sperm concentration × ml of semen) in the ejaculate. Normozoospermia, oligozoospermia, and azoospermia are diagnosed based upon total sperm count. o Normozoospermia is a term used for a completely normal semen sample. o Oligozoospermia (also often called oligospermia) refers to seminal plasma concentration < 15 million/ml. This finding can accompany a variety of defects and has implications for the type of assisted reproductive options that can be utilized, as there are significant reductions in pregnancy rates. o Azoospermia refers to the absence of sperm in the seminal plasma. Prior to the diagnosis of azoospermia, the sample should be centrifuged and the pellet examined for the presence of sperm. Types: ❑Obstructive azoospermia: This type of azoospermia means that there is a blockage or missing connection in the epididymis, vas deferens, or elsewhere along the reproductive tract. ❑Nonobstructive azoospermia: This type of azoospermia means poor or no sperm production due to defects in the structure or function of the testicles or other causes. Motility (40%) ▪ The efficient passage of spermatozoa through cervical mucus is dependent on rapid-progressive motility, that is, spermatozoa must have a forward progression of a minimum of 25 μm/s. ▪ Reduced sperm motility can be a symptom of disorders related to male accessory sex gland secretion and the sequential emptying of these glands. Motility ▪ Rapid and slow-progressive motility is calculated by the speed at which sperm moves with flagellar movement in a given volume as a percentage (range 0–100 %) by counting 200 sperms and classified as follows: A. (Rapid progressive motility: > 25 μm/s at 37 °C and > 20 μm/s at 20 °C Note: 25 μm is approximately equal to 5 head lengths or half a tail length). B. Slow or sluggish progressive motility C.Nonprogressive motility D. Immotile Motility A normal semen analysis must contain at least 32 % grade A and B progressively motile spermatozoa. If greater than 50 % of sperms are immotile, then the sperms should be checked for viability. Persistent poor motility is a good predictor of failure in fertilization, an outcome that is actually more important when making decisions regarding a couple’s treatment options. Asthenozoospermia: is used to describe the condition where a man’s semen sample shows reduced sperm motility. Causes of Asthenozoospermia Varicocele Testicular problems Poor nutrition Smoking Anti-sperm antibodies Age Exposure to toxins Cancer treatments Use of alcohol, tobacco and coffee Febrile episodes Heat exposure like in sauna, automobiles Vitality 58% Supravital staining differentiates between live and dead sperm and is assessed when sperm motility is < 40 %. ▪ A large proportion of vital, but immotile, sperm may indicate structural defects in the sperm tail. ▪ A high percentage of immotile, nonviable (dead) sperm may indicate epididymal pathology. Antisperm antibodies (ASA) may also be present, if the immotile sperms are dead. Necrozoospermia is a condition where the sperm in a fresh sample is dead or necrotic. o Normal: If fewer than 30% sperms in the sample are necrotic o Moderate Necrozoospermia: When there is 50 to 80% necrotic sperm. o Severe Necrozoospermia: If more than 80% of the sperms are necrotic. o Complete Necrozoospermia: is comparatively rare, and 0.2 to.5% of infertile men may suffer from it. Morphology 4% The staining of a seminal smear (Papanicolaou Giemsa, Shorr, and Diff- Quik) allows the quantitative evaluation of normal and abnormal sperm morphological forms in an ejaculate. Smears can be scored for morphology using the (WHO) classification. WHO method classifies abnormally shaped spermatozoa into specific categories based on specific head, tail, and mid-piece abnormalities, which is based on the appearance of sperm recovered from postcoital cervical mucus or from the surface of zona pellucida. o Teratozoospermia is a condition where there is an increase in abnormal sperms in a man’s semen. o When the concentration of sperm with abnormal morphology exceeds 96% of the semen sample, Teratozoospermia is diagnosed. o As the abnormal sperms become unable to fertilize the egg, the condition directly affects fertility.  Fructose test Fructose test in semen is a marker for seminal vesicles function and is usually performed to localize the level of obstructive azoospermia in men with low ejaculate volume. The concentration of fructose can be evaluated by a biochemical assay, which is based on the ability of fructose to form a colored complex with the indole. The lower reference limit for fructose defined by the WHO is 13 μmol per ejaculate. A low content of fructose in seminal plasma together with a low semen volume and low pH supports the diagnosis of obstructive azoospermia. Low fructose is also characteristic of CBAVD or in men with partial retrograde ejaculation.  Endtz Test Granulocytes are the most abundant type of leukocytes in human ejaculates and neutrophils are the most common subtype. Endtz test is a simple histochemical test used to detect peroxidase within neutrophils, thus allowing the screening of granulocytes in the semen. Peroxidase present in the granulocytes oxidizes benzidine derivative which precipitates to give a brown color. This test should be performed when an abnormal concentration of round cells is detected (>1 × 106 /mL).  Endtz Test However, Endtz test detects neither peroxidase-free leukocytes (e.g., lymphocytes, macrophages, or monocytes) nor those granulocytes that have already released their granules. A concentration of leukocytes ≥0.2 × 106 /mL in a semen sample is generally considered Endtz positive. The incidence of leukocytospermia in infertile men varies between 3 and 23% and has been correlated with clinical and subclinical genital infections, elevated anti-sperm antibody levels, and high levels of reactive oxygen species (ROS).  Testosterone Testosterone is excreted into the circulation, the majority of testosterone is bound to plasma proteins. The primary proteins that bind testosterone are sex hormone- binding globulin (SHBG) and albumin. The majority of testosterone is bound to albumin (54–68 %); slightly less is bound to SHBG (30–44 %). Only 0.5–3 % remains unbound or as free testosterone.  Testosterone Testosterone level (total) in adults 19+ years old At 19 or older, men should have a testosterone level of 300 to 1,000 ng/dL. Testosterone level vary daily from morning to evening so, check total testosterone between 8 a.m. to 10 a.m. Testosterone levels tend to decline by approximately 1% per year after age 35. Hypogonadism It is most commonly defined biochemically as a serum testosterone less than 300 ng/dl. Physical signs ✓ increased body fat ✓ reduced muscle bulk and strength ✓ low bone mineral density ✓ loss of body hair and a decreased need for shaving ✓ decreased energy or motivation ✓ depressed mood, decreased libido ✓ diminished work performance ✓ sleep disturbance  Prolactin In males, serum prolactin levels range from 2 to 18 ng/ml. Mildly elevated prolactin levels are not thought to cause infertility. Significantly elevated prolactin levels can inhibit the release of FSH & LH from pituitary gland and decrease T level. Assessment of Prooxidant-Antioxidant Semen Profile Reactive Oxygen Species ROS refers to by-products of the normal metabolism of oxygen. They can be free radicals, which are unstable and highly reactive compounds due to the presence of at least one unpaired electron in an outer orbital. Examples of free radicals in biological systems include superoxide anion (O2 -) and hydroxyl radical ( OH). Methods to Measure Reactive Oxygen Species Several methods can be used to evaluate ROS generation in semen or specifically in spermatozoa. The short half-lives of ROS constitute the major obstacle in the direct measurement of ROS. 2 methods used: Chemiluminescence Fluorescence: Flow Cytometry  Antioxidant Capacity Spermatozoa are then dependent on the protection provided by epididymal fluid and seminal plasma during their maturation processes. These fluids are endowed with ✓ enzymatic antioxidants, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione transferases, peroxiredoxins, and thioredoxins, and ✓ non-enzymatic antioxidants, including vitamin C (ascorbic acid), vitamin E (tocopherol), and zinc. The non-enzymatic antioxidants constitute the main defense system to scavenge excessive ROS as they account for approximately 65% of the total antioxidant capacity (TAC) of the seminal plasma.  Evaluation of Sperm DNA Damage The maintenance of sperm DNA integrity is vital for fertilization and to transmit the genetic material to the offspring. DNA damage can occur not only during spermatogenesis but also during epididymal transit or even when spermatozoa are collected for ART purposes. DNA damage refers to defects in DNA structure, which include single or double DNA strand breaks, deletion or modification of DNA bases, interstrand or intrastrand DNA cross-linkage, and protamine mispackaging. Single- (ss) or double- stranded breaks (ds) are commonly termed DNA fragmentation. Fertilization with such defective sperm carrying DNA damages may result in pregnancy loss or birth of offsprings with major or minor congenital malformations.  The Comet assay The Comet assay or single-cell gel electrophoresis is one of the simplest methods to measure single- and double-strand breaks in sperm. The principle of the assay is based on the separation of broken DNA strands under the influence of an electric field facilitated by the charge and size of the strands that can be stained with a fluorescent dye. DNA breaks have different mobility when placed in an electrophoretic field depending on the relative size of the fragment. When the electric field is applied, all the broken strands of DNA (negative charge) will migrate toward the positively charged anode, forming a comet tail.  The Comet assay Spermatozoa with more DNA breaks will show the most intense and largest comet tail After separation, the intact DNA remains in the comet’s head, whereas single- and double-stranded broken DNA fragments migrate into the comet’s tail. Comet assay: human spermatozoa stained with ethidium bromide and observed under fluorescent microscope Toluidine blue staining o This microscopy assay assesses the integrity of the chromatin DNA of the spermatozoa. It stains the damaged chromatin nuclear structure of the spermatozoa and the degree of damage is visualized by optical microscopy. o Firstly, a thin smear is prepared with the semen sample and air dried. The smear is fixed in 96% ethanol-acetone solution of equal ratio for 30 min at 4 °C. Slides are treated with 0.1 M HCl for 5 min at 4 °C and stained with 0.05% toluidine blue stain for 10 min. o Under light microscopic evaluation, a total of 300 spermatozoa were counted in different areas of each slide using oil immersion with ×1,000 magnification. o Sperm cell heads with good chromatin integrity were light blue; those of diminished integrity were deep violet (purple). Deep violet sperms were considered to be abnormal, and the percentage of sperms with a deep violet color was determined. o It is a rapid and simple assay. Sperm chromatin structure assessed by toluidine blue staining. Sperm cell heads with good chromatin structure were light blue; those of abnormal chromatin structure were deep violet. Chromomycin A3 (CMA3) staining CMA3 staining determines the damage to the DNA by measuring its protamination state. CMA3 binds more to the sperm DNA deficient of protamines, which is an indicator of poor DNA packing and damage. A semen sample smear is made on a glass slide, air dried, and fixed in glacial acetic acid–methanol (1:3) solution for 20 min at 4 °C. The stain containing 0.25 mg/mL of CMA3 with 10 mmol/L of MgCl2 is used for staining the spermatozoa. Stained slides are incubated overnight at 4 °C and examined for the presence of DNA damage. Spermatozoa with low protamination stain light yellow, whereas a bright yellow stain indicates high DNA damage. A value of >30% DNA damage for semen samples determined by CMA3 assay has a significant effect in lowering fertilization rates in ICSI.  Assessment of Sperm Fertilization Capacity For the spermatozoon to be able to fertilize, it needs to acquire fertilization potential during maturation through the epididymis. However, the ability to fertilize is only activated physiologically when in contact with the female gamete, or it can be induced in vitro using proper media. Sperm function testing also includes the evaluation of sperm mitochondrial function and fertilization events such as hyperactivation, capacitation, and acrosome reaction. Even an apparently normal spermatozoon with normal motility and morphology can be dysfunctional and not be able to fertilize.  Sperm Mitochondrial Activity Mitochondria are fundamental for human spermatozoa motility and fertilizing ability. Mitochondria participate not only in ATP production, but also in reactive oxygen species production, redox equilibrium, and calcium regulation, all of which are central for human spermatozoa motility, capacitation, acrosome reaction, and ultimately, oocyte fertilization. Mitochondrial membrane potential is a key indicator of mitochondrial health and activity. The sperm mitochondrial membrane potential (MMP) is usually evaluated using the JC-1 dye.  Sperm Mitochondrial Activity JC-1 is a sensitive marker for mitochondrial membrane potential, exhibiting a potential-dependent accumulation in the mitochondria. At high mitochondrial membrane potential, JC-1 forms J-aggregates, inside the mitochondria which emit red fluorescence, whereas at low mitochondrial membrane potential (healthy sperm), JC-1 remains at its monomer state in the cytosol , which emits green fluorescence. The calculation of the JC-1 ratio (indicative of the J- aggregates/monomers ratio) is then used to quantitatively evaluate mitochondrial health and activity.  Sperm selection o In vivo, sperm are separated and selected by different screening barriers such as cervical mucus, cumulus and zona pellucida to prevent insemination of defective sperm. o Of note, during in vitro fertilization (IVF), zona pellucida remains as the only barrier that may prevent penetration of defective sperm into oocyte, and thereby through this selection, it may increase the chance of early embryo development and pregnancy outcome. o However, during intracytoplasmic sperm injection (ICSI), even this barrier is bypassed and the only selection process that is implemented by the embryologist is based on sperm viability and morphology.  Hypo-osmotic swelling test (HOST) When the sperm sample is retrieved by testicular aspiration (TESE) or epididymal aspiration (MESA), the spermatozoa might appear immotile due to the lack of complete maturation that takes place in the final tract of the epididymis The hypo-osmotic swelling test (HOST) assumes that the tails of viable spermatozoa swell and bend if they are introduced into a hypoosmotic environment, due to the activity of the osmo-sensitive calcium membrane channels. Some studies suggest that the use of the HOST is associated with a low fertilization rate, and non-viability of sperm after 30 min of incubation in hypo- osmotic solution.  Selecting the right spermatozoon to inject ▪ Polyvinylpyrrolidone (PVP) is the most commonly used medium for immobilization of spermatozoa for ICSI. ▪ PVP media has very high viscosity that decreases sperm motility which helps selection, immobilization, and handling of spermatozoa before injection. Disadvantages of PVP PVP exposure seems to lead to lower fertilization rates as it damages the mitochondria structure and sperm membranes. ❑ Physical Examination Breast The breast examination in the fertility evaluation should focus on symmetry of the breasts and any evidence of galactorrhea as this could be indicative of a pituitary lesion. Galactorrhea is defined as active secretion of breast milk at a physiologically inappropriate time, namely other than during pregnancy or lactation. Secretions are usually white in color and occur bilaterally from hormonal stimulation of multiple ducts. Conversely, pathological discharge usually originates from a single duct and therefore is unilateral. ❑ Hysterosalpingogram o The hysterosalpingogram (HSG) test is designed to confirm that both fallopian tubes are open and the uterine cavity is normal. o The HSG is usually scheduled between days 6 and 13 of the cycle – after bleeding and before ovulation. o A HSG test may only be recommended for women with long standing primary infertility or those who have had multiple abdominal surgeries, which can cause scar tissue and fallopian tube blockages. o This test is used to diagnose intrauterine adhesions and other intracavitary defects such as polyps and fibroids as well as Müllerian anomalies such as a septate or bicornuate uterus. Furthermore, hysterosalpingography can assess tubal patency and identify the site of obstruction if the tubes are blocked. ❑ Antral Follicle Count (recruited follicles) ✓ The antral follicle count utilizes transvaginal ultrasound in the early follicular phase (cycle days 2–5) to determine the number of follicles from both ovaries(recruited under the effect of FSH) between 2 and 10 mm in diameter. ✓ Normal number 15-20 follicles. ✓ A low AFC is defined as fewer than 6 antral follicles has been shown to correlate with poor ovarian reserve serves as a good predictor of poor response to ovarian stimulation and, to a much lesser extent, of poor oocyte quality and nonpregnancy. ❑ Folliculometry (Test of Ovulation) Folliculometry is a procedure that involves performing a series of ultrasound examinations in order to monitor the growth of follicles in the ovary. It is done from the 9th to the 20th day of the menstrual cycle (if the cycle lasts 28 days). The follicle grows 1-2 mm per day, and the ovulation is ready when it is in the diameter of 17-25 mm. During folliculometry, the number of follicles in development are monitored and recorded, their size and the time of ovulation. Chlamydia Antibody Testing o Salpingitis accounts for greater than 50% of cases of tubal factor infertility. o Tubal pathology is one of the causes of subfertility being responsible for 10% to 30% of the cases in developed countries and up to 85% in developing countries. o Tubal pathology and infertility have been associated with asymptomatic Chlamydia trachomatis infections, chlamydia antibody testing (CAT) in serum has been introduced as a screening method for tubal factor subfertility. o Infection with Chlamydia trachomatis stimulates humoral response and synthesis of IgG, IgA and IgM antibodies by plasmatic cells. o Progressive increase in serum concentrations of specific IgM, IgA and IgG is typically observed between 5–20 days of infection. o IgM, a marker of acute infection, can be detected about the day 5, and elevated levels of IgA are usually observed after 10 days. o IgG are usually detected at 2–3 weeks of the primary infection. o Reinfection is associated with a rapid increase in IgG titer which remains elevated for weeks and then gradually decreases Biochemical Markers of Ovarian Reserve Time- FSH- Estradiol- Inhibin B (Day 3) limited Progesterone (Day 21) Non time- limited AMH – TSH – Prolactin - oxytocin Serum Progesterone (Day 21) ▪ Measurement of serum progesterone levels can also be used to document ovulation. ▪ Serum progesterone levels remain below 1 ng/mL during most of the follicular phase rising during the late follicular phase to 1– 2 ng/mL. ▪ After ovulation, progesterone is secreted from the corpus luteum and levels rise steadily until they peak approximately 7–8 days following ovulation.. Serum Progesterone (Day 21) ▪ Typically, a serum progesterone level >3 ng/mL provides reliable evidence that ovulation has taken place but does not provide information on when it occurred. Classically, it can be measured on day 21 with the assumption being that the woman has a 28-day menstrual cycle. ▪ If a woman is able to estimate how long her menstrual cycles are, then she can simply obtain this test approximately 1 week before her period is due. Anti-Müllerian Hormone ✓ AMH, secreted by the granulosa cells of the growing follicles, is a reflection of the primordial follicle pool. ✓ It is a convenient test, because it can be measured at any time in the menstrual cycle. ✓ This hormone is also used as a marker for recovery of ovarian function after completing chemotherapy/radiotherapy treatments or as a marker of decreased ovarian function in women that choose not to preserve fertility and hope to maintain their ovarian function in follow-up visit. ✓ Although exact cut-offs for good versus poor ovarian reserve have not been established, lower levels (< 1 ng/ml) have been correlated with poor ovarian response. Oocyte meiotic maturation The maturation of oocytes includes two interrelated processes: nucleus and cytoplasmic maturation. Human oocytes are arrested within ovarian follicles at the diplotene stage of the first meiotic prophase (a germinal vesicle stage) until puberty when the ovarian cycle begins. Following a surge in luteinizing hormone (LH), the oocyte reinitiates meiosis. The first visible sign of oocyte meiotic maturation is breakdown of the oocyte nuclear membrane referred to as germinal vesicle breakdown (GVBD).  This is followed by chromosome condensation and alignment of the chromosomes at the metaphase plate at metaphase I. These oocytes are referred to as metaphase I (MI) oocytes. This is followed by the first oocyte meiotic division, extrusion of the first polar body and formation of a secondary oocyte (mature egg). The first polar subsequently positions itself within the perivitelline space. The second meiotic division thus stops at the in metaphase II. Secondary oocyte (mature egg) that arrests at metaphase II until fertilization when the second meiotic division is completed. Oocytes retrieved from patients following controlled ovarian hyperstimulation show varying stages of meiotic maturity. Only oocytes at metaphase II (MII; oocytes with first polar body extrusion) are suitable for ICSI procedures. Oocytes at metaphase I (MI; first polar body not extruded) and at prophase I (showing a germinal vesicle) cannot be used for ICSI because they have the diploid chromosomal set. These oocytes are placed in the maturation medium for extrusion of first polar body and cytoplasm maturation,  Morphological determination of oocyte quality The oocyte quality can be determined more specifically by evaluation of the peculiarities of the following: o Oocyte cumulus–corona-complex (OCCC) structure o Oocyte cytoplasm o Dimension of the perivitelline space o Zona pellucida o Polar body o Meiotic spindle Oocyte cumulus–corona-complex (OCCC) structure ▪ During follicular antrum formation, granulosa cells (GCs) differentiate into: ✓ mural GCs, lining the follicular wall, and ✓ CCs, surrounding the oocyte. ▪ Within the cumulus mass, CCs in close contact with the oocyte (corona cells) develop cytoplasmic projections which cross the ZP and form gap junctions with the oolemma. This organized structure is called the cumulus –oocyte complex. ▪ In natural spontaneous cycles, oocyte nuclear maturation runs parallel to the gradual FSH-dependent expansion of the cumulus and corona cells. Oocyte cumulus–corona-complex (OCCC) structure ▪ The OCCC collected from ovarian follicles differ in the texture and compactness of the cumulus and characteristics of oocyte cytoplasm. ▪ The evaluation of oocyte maturity has been based on the expansion of the cumulus surrounding the collected oocyte. ▪ Cumulus expansion is the formation of the HA-rich extracellular matrix. ▪ Cumulus expansion is essential for ovulation and fertilization and is predictive of oocyte quality. Oocyte cumulus–corona-complex (OCCC) structure ▪ Using this approach, embryologists may categorize oocytes as metaphase II when showing an expanded cumulus and the characteristic `sun-burst' appearance of the corona radiata. ▪ A less expanded OCCC may be suggestive of intermediate maturity while the absence of an expanded OCCC may be suggestive of immaturity  Perivitelline space and zona pellucida The perivitelline space (PVS) is a “space” between the surface of the oocyte or more specifically the oolemma and the zona pellucida, an extracellular matrix synthesized by the oocyte. The PVS has contents that change during development and that appear to play various roles before, during, and after fertilization.  Perivitelline space and zona pellucida The PVS varies considerably in size at different times in development. The PVS around germinal vesicle containing oocytes is relatively small and difficult to visualize with light microscopy. After extrusion of the first polar body, the PVS becomes asymmetrical and enlarged around the first polar body, and it retains this appearance at the time of fertilization. After fertilization, the PVS remains asymmetrical and large around the polar bodies. As cleavage occurs, the PVS takes on a different shape as it follows the contours of the blastomeres  Perivitelline space and zona pellucida Formation of the Zona pellucida composes of three layers. Total thickness of zona pellucida varies from 10 to 31 µm (normally 15-20 µm). Immature human oocytes have thicker zona pellucida (20.4±2.4 µm) than mature oocytes (19.5±2.2 µm) and day 3 embryos (15.2±2.8 µm). Zona pellucida thickness influences sperm penetration. The highest fertilization rate of oocytes was when the thickness of the zona pellucida was less than 18.6 µm Metaphase II oocyte observed by polarized light microscopy. MS meiotic spindle; IL zona pellucida inner layer; ML zona pellucida middle layer; OU zona pellucida outer layer  First polar body Morphology of the first polar body indicates the postovulatory age of the human oocyte. The degeneration and deviation of the first polar body from the metaphase II spindle show oocyte aging. Some morphological criteria of first polar body such as: ✓ the shape (round or ovoid), ✓ size (large or small), ✓ surface (smooth or rough) and ✓ integrity of cytoplasm (intact or fragmented) can be used to predict the oocyte quality.